Applichem Immunoassay Buffer

Immunoassay Buffer
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Immunoassay Buffer
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Die moderne Gentechnik zeigt, dass in vielen Fllen schon freie DNA-Molekle fr Infektionen, Rekombin ationen oder biologische Transformationen ausreiche n [1,2]. Zustzlich werden die Nachweisverfahren fr DNA-Molekle immer sensitiver. Daher wird die Detektion von Kontaminationen oder die Verhinderung von Amplifikations-Artefakten in der PCR fr die Gentechn ik, die Kriminalistik, die Biomedizin und die Hygiene immer wichtiger. Die vollstndi ge Dekontamination von Gerten und Materialie n von DNA-Moleklen wird so zu einem entscheidenden Faktor fr die allgemeine biologische Sicherheit.
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Keywords NukleinsureDekontamination DNA-Degradationstest PCR-Test
A comprehensive catalogue of products, with detailed

Antibody Stabilisation Autoklavieren von DNA ELISA Plates Cross-reactivity Interfering effects
information provided by no other catalogue in the field, is available in German and English.
Alles oder Nichts: Erstaunlich e Erkenntnis Das Mittel der Wahl zur Beseitigung von Kontamina Kontaminationen durch Nukleinsuren ist immer noch Chlorbleichlauge (bleach) ein Mittel das alles zerstrt, nicht nur die Nukleinsure. Dies hat uns veranlasst in Kooperation mit multiBIND Biotech, Kln, nach einer unschdlichen Alternative zu suchen und die molekulare Wirkungsweise sonstigen DNA-Dekontaminationsmittel der auf dem Markt befindlichen zu untersuchen. Hierfr wurde unter suchen. sehr hoher Belastung (groer DNA-berschuss) mit definierten DNA-Kontami DNA-ber nationen die Eigenschaften der konventionelle n Mittel verglichen. Zwei Probleme werden offensichtlich: Erstens werden durch die konventionellen Mittel in keinem Fall die DNA-Molekle effizient zerstrt und zweitens enthalten diese Mittel Komponenten mit stark korrosiven oder giftigen Eigenschaften fr uns die Notwendigkeit der Neuentwicklun . Als Fazit daraus hat sich g einer effektiven Lsung zur DNA-Dekontam DNA-ExitusPlus und Autoclave-Exit ination ergeben, die wir hier als usPlus vorstellen. Im Vergleich zu den herkmmlichen Produkten wird RNA schnell und effizient zerstrt, ohne DNA und dass das Reagenz korrosive oder giftige Eigenschaften aufweist. Bei der DNA-Dekontamination unterscheidet man nach der molekularen Wirkungsweise der eingesetzten Mittel drei Grundprinzipien zur Zerstrung oder Inaktivierung der genetischen Information: Modifikation, Modifika Je nach Zusammensetzung der Mittel Denaturierung und Degradation. knnen diese drei Prinzipien einzeln oder in Kombination angewandt werden. Da nach den aktuellen Erkenntnissen zum biologischen Risikopotenzial von freien DNA-Moleklen fr eine wirklich DNA-Dekontamination die Zerlegung sichere dieser DNA-Molekle in mglichst kleine Fragmente die wirkungsvollste Methode wurden die gngigen konventionellen tionellen Mittel mit unserer Neuentwicklun ist, g DNA-ExitusPlus im DNA-Degradat glichen. Der DNA-Degradationstest erlaubt ionstest verver einen sensitiven, quantitativen Vergleich der Geschwindigkeit des DNA-Abbaus (Abb. 1 und 2). Unerwarteter Weise haben wir festgestellt, dass einige der bekannten kommerziellen Mittel nur mit dem Prinzip der Modifikation oder Denaturierung der DNA-Molekle arbeiten. Eine Zerlegung der DNA-Strnge genetische Information, fr die diese erfolgt dabei nicht, sondern die Informa DNA-Strnge kodieren, wird eigentlich nur maskiert. Eine chemische Demaskierung der DNA-Molekle durch Entfernung der blockierenden Gruppen wrde die genetische Information wieder lesbar plifizierbar machen. Nach dem heutigen und amWissensstand zur Gentechnik und der Problematik der Neukombination von trgern sind solche Mittel eigentlich nicht Erbmehr zeitgem. Aber auch die Mittel, die zu einer nachweisbaren Degradation
Keywords Immunoassays
Why is there such a great difference in storage between h The reason is that in professional ELISA kit production the plates are not easy to perform process has been an industry standard for thirty years. Fo coating stabiliser solution. It is just as simple as a second blocking step lutions freely available in low volumes for use in research lab until now. AppliC in volumes starting as small as 50 ml, which is called the AppliCoat Plate Stabi to-use and has a great advantage compared to almost any stabiliser used antibodies and antigens than most other products do. And there is a secon Two benefits with one solution When antibodies are coated onto ELISA plates, most of the antibodies are not into close contact to the plastics surface of the ELISA plate, conformational ch The result is that most antibodies coated on a plate are unfolded or inactive. O active and can bind to analytes and this is greatly variable depending on the s really differ from batch to batch or even from well to well. These differences from well to well can affect the variability of an assay, bec way of refolding antibodies and of preserving antibodies from conformati decrease such variabilities in assay performance. This is a key benefit of Ap coated proteins to refold and then to preserve active conformation over a lo antibody conformation of some of the coated antibodies and 2. Preserving c fits are used for production of high-quality ELISA kits as well as in resear Stabiliser the percentage of active antibodies will still be in the range of 28 from well to well and from plate to plate can be minimised in most assays depend on the used antibodies, but when ELISA are validated (e.g. according Validation, FDA, 2001) or according to other validation strategies, the differe The positive effects of AppliCoat Plate Stabiliser are shown in Fig. 1. A sandwi
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Dekontamination der Haut und Hnde von Nukleinsure n
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Freie Nukleinsuren verursach en als Kontaminationen groe Probleme im Forschungs- und molekular biologisch-analytischen oder klinisch-diagnostischen Labor. Durch die extrem hohe Sensitivitt von DNA-Nach weistests, knnen kleinste Verunreinigungen in PCR-Ans tzen zustzliche Arbeit bedeuten und im schlimmsten Fall Ergebnisse verflsch en. Mit Derma-ExitusPlus (HHDK) aus der Serie von ExitusPlus-Produkten wird erstmals ein vllig neuer Anwendungsbereich erschlossen bzw. zustzlich e Kontaminationsquellen ausgeschlossen.
Size-Exclusion Chromato grap for purification of biomolec hy ules
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Size-exclusion chromatog raphy (SEC) is a popular method to separate biomolecules based on their size. Primarily, it is applied to the separation of biopolymers such as proteins and nucleic acids, i.e. water-soluble polymers. This system is also called gel filtration, typically with beads of dextran or agarose serving as gel matrix. Smaller molecules pass significant ly slower through the column than larger molecules. Not to be mixed up with gel electropho resis, there are big difference s in terms of the separation principle. SEC does not require electric current and the sieving effect will not separate small molecules first.
DNA-freie Reagenzien und Mastermixe fr die PCR
Der Anteil von molekular biologischen Nachweismethoden ist in den letzten Jahren erheblich gestiegen, besonder s in den Bereichen Qualittsk ontrolle, Forensik, klinischer Forschung und Diagnostik insbesondere der Infektionsdiagnostik . Gerade fr diese Applikatio nen werden hochsensitive und gleichzeitig zuverlss ige PCR-Tests bentigt. Dafr bietet AppliChem nun optimiert e PCR-Kits an und widmet sich explizit der Hintergru ndproblematik, die durch DNA belastete Reagenzie n und Arbeitspltze entstehen kann.
ine der Hauptquellen fr Kontamination en mit Nukleinsuren ist der Experimentato r selbst. Die Nukleinsuren stammen B. aus Hautschuppen, Haaren und Speichel oder von Mikroorganismen, die seine Haut besiedeln oder z.B. beim Niesen eigesetzt werden. Gelangen diese in die PCR-Anstze oder PCR-Reagenzi en, knnen s
Keywords
It is indeed correct that smaller molecules
pass more slowly through t
contents
Limiting Cross Reactivity in Immunoassays Without Blocking .... No Result! Tips for storing antibodies Comparing Blocking Reagents Improving quality & stability of ELISA Products with application notes Antibody Stabilizer-PBS Antibody Stabilizer-Tris Applicoat Plate Stabilizer Blocking Buffer I Blocking Buffer II EGrade Blocking Buffer III BSA Blocking Reagent CA Coating Buffer pH 9,6 Coating Buffer pH 7,4 CrossDown Buffer CrossDown Peroxidase-Stabilizer Peroxidase-Stabilizer Sample Buffer TSample Buffer T+ Stripping Buffer I Washing Buffer TrisT- (10X) Washing Buffer TrisT+ (10X) Related products Further reading 21 21 22 23 24 25 26 27 27 28 31 32 33 33 34 34 35 36 36 2 10 12 14 18
2010 AppliChem Immunoassay Buffer
immunoassay
Limiting Cross Reactivity in Immunoassays

Antibodies are used in Immunoassays to easily and specifically distinguish between different substances.
Dr. Wolfram H. Marx, AppliChem; PD Dr. Wiesmann, University of Mnster; Dipl. Biol. Susanne Siewert, University of Ulm; Dipl. Chem. Nico Dankbar, University of Mnster; Dr. Peter Rauch, CANDOR Biosciennce GmbH; Dr. Christoph Specht PARA, Bioscience GmbH There are several types of these assays including Enzyme linked immunosorbent assays (ELISA), enzyme immunoassays (EIA), Western blotting, radioactive labelled immunoassays (RIA), protein arrays, immuno histochemistry, and immunopolymerasechain reaction (ImmunoPCR). Each of these assays have one drawback in common cross reactivity. Immunoassays are a very important tool in bioanaly tical and biochemical laboratories. They are used in research, food and environmental monitoring as well as in diagnostic applications. Immunoassays are quite easy to carry out and very specific in terms of quanti tative and qualitative significance due to their use of antibodies for detection. Theoretically, each antibody can identify one antigen and binds this antigen with high affinity which explains why one can distinguish so easily between different substances. In practice, it is not that simple. Immunoassays suffer from cross reactivity which results in false bands in Western blots, signals in the negative control of an ELISA or a very high background in a protein array. Every false result means more work, additional costs and potentially misdiagnosis of patients [1]. Although antibodies are very specific and have high affinity for one antigen in particular, often antibodies can also bind with lower affinity to other antigens which are not detected by the assay. This is even observed with very
labeled detection antibody analyte capture antibody heterophilic antibodies or HAMA blocking protein of surface interfering or cross reacting substance
Fig. 1 The perfection: interference-free sandwich assay. Fig. 2 A on-specific binding of a labeled detection N antibody to a not (sufficiently) blocked surface. Result: false-positive signal. B on-specific binding of a labeled detection N a ntibody to a blocked surface. Despite blocking of the surface the antibody binds to the blocking protein itself. Result: false-positive signal. C n interfering protein binds to the Fc segment A of the detection antibody and hinders sterically the binding of the analyte. Result: false-negative signal. D he capture antibody binds to the Fc segment of T the labeled detection antibody. The analyte cannot be bound by the capture antibody any more. Result: false-positive signals
Immunoassay Buffer AppliChem 2010
well characterised antibodies known to have a high affinity to the target analyte. The result are interferences such as nonspecific binding, cross reactivities and matrix effects leading to high background with bad signaltonoise ratio.
Non-specific binding
Nonspecific binding occurs when an antibody binds to substances present in much higher concentrations than the target analyte (e.g. nonspecific binding to albumin or immunoglobulins), binding to surfaces (e.g. Western blotting membranes or ELISA wells), or binding to loci on immobilized antibodies in protein arrays [2]. Assays with insufficient blocking or with difficult matrices con taining e.g. a high albumin concentration or high con centration of endogenous interfering substances, are strongly affected. There are also other causes of non specific binding. Since detection antibodies are labelled with enzymes (e.g. alkaline Phosphatase or Peroxidase), fluorescent dyes, radioactive isotopes or DNA (Immuno PCR), the label itself can also be a source of unwanted interactions. In the case of fluorescent dyes, which are frequently hydrophobic, the binding properties of detection anti bodies can be changed. The dyes themselves may cause unwanted binding and thereby reduce the solu bility of the labelled protein. The antigenantibody bin ding can be impaired too [3]. These effects may lead to increased nonspecific binding of the labelled antibody onto surfaces (fig. 2A and 2B), to foreign proteins of the real sample (fig. 2C) or to the capture antibody (fig. 2D). In those cases false positive results are obtai ned even in the absence of an analyte or the whole assay shows a high background, respectively. In protein arrays this phenomenon leads to higher background fluorescence of single spots or a low signaltonoise
Interferences in immunoassays
Interference can come in several forms such as cross reactivities, nonspecific binding and matrix effects. Laboratory or clinical samples may contain foreign sub stances in concentrations that can interact with the ana lyte or the capture/detection antibodies thereby dis rupting the desired reaction. Similarly, surfaces that act as platforms for immunoassays have also been known to be a source of interference. By applying novel buf fers (e.g. CrossDown Buffer), most of the above men tioned effects can be avoided. Simply exchange the sample buffer or antibody dilution buffer by CrossDown Buffer and thereby improve the quality of the assays and the efficiency of the assay development. To understand the basic principles of interference, we first take a look at an ideal sandwich ELISA (fig. 1) followed by fig. 25 where interference is demon strated. In a troublefree sandwich ELISA, the capture antibody is immobilised on the bottom of a well. The rest of the surface is blocked sufficiently. The capture antibody binds the analyte, while the secondary labelled antibody, binds to a different site on the analyte (fig. 1).
Fig. 3 I ross reactivity of an interfering substance with C the capture antibody. Result: false-negative signal. J ross reactivity of an interfering substance with C the detection antibody. Result: false-negative signal. K ross reactivity both with the capture and with C the detection antibody. Result: false-positive signals Such a phenomenon is rather seldom in practice, but definitely possible with antibodies having lower specificity. Such an interference picture may occur with antibodies directed to a target with a conserved amino acid sequence of a protein whose sequence motive also occurs in other proteins.
Fig. 4 Fig. 5 F ridging by HAMAs and heterophilic antibodies, L asking of the analyte by a protein of the B M respectively, resulting in a coupling of the specimen. The epitope is blocked for binding capture and detection antibody. of the capture antibody, resulting in no binding Result: false-positive signals. to the analyte at all or in the case of a sterical G n anti-idiotypic HAMA binding to the capture A hindrance binding is very week. antibody. The interfering antibody binds in the Result: false-negative signal. area of the highly variable region of the Fab segment and thus prevents the binding of the analyte. Result: false-negative signal. H n anti-idiotypic HAMA binding to the detection A antibody. The interfering antibody binds in the area of the highly variable region of the Fab segment and prevents the binding of the analyte. Result: false-negative signal.
immunoassay
ratio, respectively. Fluorescent dyes also may bind pro teins or antibodies from serum samples, resulting in a reduction of the dye fluorescence and in extreme cases to a complete quenching of the signal. Based on this, discussions are ongoing to eliminate fluorescent dyes from protein arrays completely [4]. The complexity of protein arrays is very high due to the application of many different capture antibodies and labelled detec tion antibodies in one reaction. Thereby, the risk of nonspecific binding of proteins in the sample or labelled antibodies to single spots increases significantly as well as interferences of components of the sample with the antibodies [2].
speaks about a matrix effect. There is a smooth tran sition to all other negative effects. Matrix effects can be caused by AntiAnimalAntibodies, heterophilic anti bodies, endogenous interfering substances or influ ences like viscosity, pH or salt concentration. There are negative effects that are restricted to medical and diagnostics assays. These effects are based on interfering substances present in human specimen like plasma, serum or tissue samples. Since the results of these assays are the basis of patient therapies, inter ferences and false results are severe.
Anti-Animal-Antibodies
Human AntiAnimalAntibodies (HAAA) are of the IgG, IgA, IgM or IgE type and are formed as an immune response after contact with animal immunglobulins. HAAAs are well known from diagnostic assays. Studies report that up to 80 % of all samples contain HAAAs. The concentrations can be very high, reaching levels of several milligrams per milliliter [7]. HumanAntiMouse Antibodies (HAMA) are the best known interfering antibodies in immunoassays. HAMAs are human antibodies which are relatively specific and which can bind mouse antibodies with a middle affinity up to, in some rare cases, a high affinity. One of the reasons for the development of these antibodies can be nonhuman therapeutic antibodies which are admin istered as drugs (e.g. in cancer therapies). After thera peutic medication the human immune system reacts to these foreign antibodies and begins to produce anti bodies against these mouse antibodies. Therefore, HAMAs interfere with immunological methods which include mouse antibodies. In sandwich ELISAs based on monoclonal mouse antibodies, capture and detection antibody will be bridged (fig. 4F), resulting in a false
Cross reactivity
Cross reactivities are the result of nontarget binding and appears similar to nonspecific binding. Unlike nonspecific binding, however, when one talks about cross reactivity the cross reacting substance is known and its cross reacting properties can be proven e.g. by measurement of the competing concentration of the cross reacting species [5]. Cross reactivity is the ability of the antibody to bind structures other than the target analyte (fig. 3I, 3J and 3K). Often, these structures are similar to the analyte such as metabolites or chemical substances with a similar molecular structure. Proteins with evolutional homology of amino acid sequence or similarity in tertiary structure can cross react too. Cross reactivities play a key role in many competitive assays, because only one antibody is used [1, 4]. For these assays, it is part of the validation to identify and to quantify possible cross reactors experimentally [5]. Cross reactivities can also play a major role in the detection of proteins in Western blots or in immuno histochemical applications. Cross reactivity can result in staining of additional bands in Westerns or cell struc tures, without knowing the exact molecular reasons for this unwanted binding. In Western blots, sometimes the additional bands simply represent protein fragments which originate from the normal degradation pro cess. But in some cases it is important to look closer at cross reactivities caused by the primary or secondary antibody. For any scientific publication it is necessary to verify the reasons for unexpected bands and signals anyway.
Matrix effects
The least defined term is the matrix effect. Matrix effects are the sum of all negative effects of all compo nents within a sample, which can affect the determina tion of the target analyte [6]. If the exact molecular cause of such an effect is unknown, but can be related to the composition of the sample to be determined, one
positive signal. Due to the sequence homology between antibodies of different species, HAMAcontaining sera may disturb assays which contain antibodies from other species. Drugs aren't the only reason for the development of HAMAs. Contact with domestic animals over several years enhance the formation of anti animal antibodies that either bind to antibodies of a single species (e.g. rabbit, mouse, dog, hamster) or binding to antibodies of several species with different affinities. Some interfering antibodies can bind to the Fc portion of the antibody, while others bind to the Fabfragment resulting in redu ced binding of the analyte or even completely preven ting the formation of any real complexes. The conse quence is a false negative measurement (fig. 4G and 4H). The ability of HAAAs to bind the Fcfragment is called antiisotypical interference. In contrast, antiidio typical interfering antibodies bind the highly variable, Fab portion of the antibody [7].
Heterophilic antibodies
According to Tabers Medical Dictionary, 'heterophilic antibodies are antibodies which bind other antigens than the specific antigen'. Heterophilic antibodies can be of the IgG, IgM, IgA or IgE type. The IgM type plays a key role in sera from rheumatic patients. These sera do contain so called rheumatic factors in high concen tration. Rheumatic factors are IgM type antibodies which bind to Fcfragments of human antibodies, and therefore they may bind to the Fcfragments of antibo dies of other species in the assays as well. Rheumatic sera lead to a linkage of capture and detection antibo dies with the consequence of falsepositive signals. This reflects the general interfering mechanism of hete rophilic antibodies. The effect of the rheumatic sera is similar to the effect of HAAAs. The difference between HAAAs and heterophilic antibodies is their formation. The latter aren't formed upon contact with animal immunglobulins, but rather they are multispecific anti bodies of the early immune response or interfering antibodies with unknown immunological origin [7]. Interference by HAAAs or by heterophilic anti bodies have been known now for more than 30 years. In general, the interfering antibodies are weakly binding antibodies [7], which predominantly disturb assays that, due to the low concentration of the analytes, require a low dilution of serum or plasma specimens [8]. Addi tion of blocking substances to the sample buffer, e.g. nonspecific sera, antibody fragments or high concen trations of animal immunoglobulins, are able to reduce the negative effects of the HAAAs or heterophilic antibo dies by competition, but dont always prevent them [7].
Interference caused by endogenous components of the specimen

Even naturally occurring proteins found in specimens can interfere with immunoassays. Some well known interfering substances in human sera are albumins, complement factors, lysozymes and fibrinogen [4]. Since analytes of low molecular weight can bind readily to albumin, this reduces the accessibility of the anti body to the analyte. Numerous hormones are bound to transport proteins, which may lead to difficulties as well. The binding ability of certain proteins is a sub stantial part of their biological function, e.g. albumin, complement and Creactive protein (CRP). Because these proteins are natural receptors for many sub stances, nonspecific binding or even cross reactivity is possible which complicates the recognition of certain analytes in an assay similar to antibodies. Endogenous proteins can bind as interfering factors to antibodies (fig. 2C, 3IK) or mask the target analyte (fig. 5L). For example, lysozyme binds nonspecifically to any proteins with a low isoelectric point. Therefore, antibodies which have 2010 AppliChem Immunoassay Buffer
immunoassay
an isoelectric point of approximately 5, can be bound and form a bridge between capture and detection anti body [4]. One other important aspect, which should be mentioned, is the interference by strongly fatty speci mens, because some analytes are fatsoluble and the binding between antibody and analyte can be affected by lipids.
Avoiding the interference by applying novel immunoassay buffers examples from the practice
In most cases, the problems in many immunoassays are caused by low to medium affinity bindings. The best known strategy to circumvent the negative effects is an optimised blocking procedure. To get the systems run ning many blocking solution were developed. Most of them can be called 'very creative', but rather lack real good results in practice. The larger the analyte, the easier is the blocking. Small analytes often require a more efficient blocking. The optimal blocking buffer shall be a generally applicable solution for most immunoassays and shall give reliable results. Only such a multipurpose solution can help saving time and money for optimizing and developing immunoassays. This holds true especially if expensive antibodies or difficulttoprepare samples are applied. Caseinbased blocking solutions have proven to be very efficient. But preparing such a solution with consistent blocking efficiency requires a great deal of time and experience. The reason is simple. Simply solubilizing casein doesnt give a good blocking solution. That kind of casein blocker is available from many suppliers, but the results in assays are not of the same quality like they should be. Literature describes and practice shows that casein works best, if it is cut into fragments of different mole cular weights. Nowadays, a chemical modification during the manufacturing process allows the produc tion of casein solutions with reproducible and reliable results. Replacement of an unsuitable blocking reagent in immunohistochemistry makes the interpretation of an experiment with osteoblast culture possible (fig. 6). On the first day, a freshly prepared osteoblast culture shows no or a very weak expression of the extracellular matrix protein osteocalcin. By applying a novel casein based blocking reagent (Blocking Buffer I, AppliChem) in combination with antiosteocalcin (monoclonal, TaKaRa), the actual expression is correctly detected (fig. 6 upper left). Standard blocking with BSA leads to a completely falsepositive result (fig. 6 upper right). With time, the cultured osteoblasts build up the extra cellular matrix and osteocalcin is synthesized. The expected increase in osteocalcin expression can be correctly monitored by using Blocking Buffer I. The
staining of the cultures intensifyes with time (fig. 6 lower left). In many cases the substitution or optimisation of the blocking reagent alone is not sufficient, because the blocking agent has limited influence on all the different negative effects. The solution to combating the one common feature of many interfering substances, i.e. low to medium affinity binding, was used to develop a new buffer. CrossDown Buffer (AppliChem) capitalizes on the fact that the binding of interfering substances is weaker than the specific binding of the target analytes. It elimi nates low and medium affinity binding, without nega tively affecting high affinity binding and high specificity. Figures 7 to 10 show different examples of typical in terference effects in immunoassays that are prevented by the use of CrossDown Buffer. Figure 7 shows a Western blot with high back ground. One of the typical daily problems in many labs. Only the substitution of the blocking reagent and additionally substitution of the antibody dilution buffer led to an analyzable result: Myostatin (GDF8; 12 kDa) from mouse myoblasts (C2C12) was blotted on nitrocellulose NC45 (Serva) and detected with anti GDF8 (Santa Cruz). Originally, blocking was perfor med with 2 % nonfat dried milk powder and 1 % BSA in TBS. As antibody dilution buffer 0.3 % BSA in TBS was applied and detection was done with ECL (Amersham). Applying the conventional protocol, the bands are hardly visible. With the substitution of Blocking Buffer I and CrossDown Buffer as new anti body dilution buffer, a significant reduction of back ground is achieved (fig. 7). Positive results in a protein chip application are shown in fig. 8. CrossDown Buffer reduced a high background and improved the signaltonoise ratio from 3.4 to 17.3. In this experiment different polyclonal anti EPIL antibodies (EPIL early placenta insulin like growth factors) were tested for their suitability. The purified antibodies were immobilized on aminosilane functionalized microarray slides using a spotter (GMS 417) at a concentration of 500 g/ml in a volume of 1.8 nl/spot. Afterwards, 2 ml supernatant of an EPILover expressing cell line (SKBR3) were mixed with the dye Oyster650P (Denovo Biolabels) and all proteins of the mixture were labeled. The incubation on the slide was carried out at a dilution of the medium : buffer 1 : 20 with CrossDown Buffer in comparison to PBS. After washing of the slides they were analyzed with a flu orescence scanner (GMS 418) and the data were evalu ated with ImaGene (Biodiscovery Inc.). The use of CrossDown Buffer resulted in a clear reduction of the background signal, allowing the selection of antibodies in terms of their suitability to detect EPIL. An example of the impact of a matrix effect on an ELISA is shown in figure 9. With this model assay a
Fig. 6 Osteoblast culture for detection of osteocalcin. Whereas blocking with BSA gives a wrong result (upper right), switching to a novel caseinb ased blocker (Blocking Buffer I, AppliChem) shows correct results (upper left). The time course of expression can be shown correctly (lower panels). (images by PD Dr. Wiesmann, University of Mnster, Germany)
Fig. 7 Western blot. Left side without and right side with Blocking Buffer I and CrossDown Buffer. Detection of myostatin in mouse myoblasts with anti-GDF-8 as primary and rabbit anti-goat IgG-HRP as secondary antibody on a nitrocellulose membrane NC45. (Dipl. Biol. S. Siewert, University of Ulm, Germany) with Standard Assay Buffer with CrossDown Buffer
Fig. 8 Reduction of a non-specific interaction of the detection antibody with the array surface by the use of CrossDown Buffer. Signal-to-noise could be increased from 3.4 to 17.3. (Dipl. Chem. N. Dankbar, University of Mnster, Germany)
immunoassay
matrix effect was induced systematically. The assay was performed by Candor Bioscience GmbH, a company, which develops and validates assays for pharmaceu tical research and diagnostic applications. Rabbit serum was used as a matrix and spiked in defined concen trations with human Creactiveprotein (CRP, Biotrend). As capture antibody, Clone C2 was used (Biotrend, 1 g/ml coating concentration in PBS) and for detection, the biotinylated antibody from Clone C6 (Biotrend, working concentration 2 g/ml) was applied. The spiked serum samples were diluted either with a PBS BSA buffer or with CrossDown Buffer 1 : 2 and mea sured by ELISA. Detection was carried out with NeutrA vidin conjugated horseradish peroxidase (Pierce, working concentration 0.05 g/ml in PBSBSA buffer) with ImmunoPureTMBsubstrate (Pierce). A matrix effect, whose exact molecular reason is not known, leads to a calibration curve with low sensi tivity. Due to its physiological function, CRP is able to bind many proteins and substances (scavenger func tion of CRP), probably causing a significant reduction in the accessability of the epitope by the antibody. Pre sumably, an interfering effect as shown in figure 5L takes
place, although interfering effects as shown in figure 3I K can not be excluded. Again, CrossDown Buffer pre vented the binding of CRP to endogenous substances of the rabbit serum and thus improved the sensitivity of the calibration curve by the factor of 3 (fig. 9). The ELISA shown in fig. 10 is an example, where the substitution of sample and antibody dilution buffer by CrossDown Buffer was sufficient to achieve a good result. As antigen a lysate of human kidney carcinoma cells were immobilized and a serial dilution of two im mun sera in repeat determination (1:50 to 1:36450) AG loaded in columns 14. The corresponding preimmun sera (1 : 50) were pipetted in lane H. Blank values are in column 5. The result of using the standard buffer (PBS/NaCl/Tween 20) in contrast to the new CrossDown Buffer is selfevident. It leads to a better sensitivity by reducing the level of detection (LOD) from 0.051 to 0.022 and the level of quantification from 0.152 to 0.065 and enlarging the measurement range. The improvement can be explained by the elimination of the falsepositive signals of the preimmun sera and the reduction of the background.
Extinction 450 nm
PBS/BSA-Standard buffer CrossDown Buffer
CRP [ng/ml]
Fig. 9 ELISA of CRP in rabbit serum (developed by P. Rauch, Candor Bioscience GmbH). CrossDown Buffer improved sensitivity by avoiding a matrix effect.
Conclusion
The phenomenon of interference in immunoassays is as old as the application of antibodies for bioanalytical and diagnostic purposes. During the last 30 years numerous molecular causes were found and the mechanism of interference investigated which led to the development of prevention strategies. At todays state of the technology, many interference effects can be minimized and innovative buffers for immunoassays make an essential contribution to it. In fact one can say, that good solutions for these problems have been developed. It is new that the same sample and anti body dilution buffer allows to minimize different inter ference effects with different molecular principles at the same time. CrossDown Buffer is applicable for dif ferent immunoassays. The results shown here cover just a part of the different negative effects in different methods, which could be minimized or even avoided with this novel buffer. In addition, nonspecific binding in immuno histochemical applications and falsepositive binding in immunoPCR can be prevented. The novel Blocking
Solution I, which is manufactured in reproducible qua lity with its wide spectrum of fragments of different molecular weights, can help to increase the efficiency of these methods. Taken together, costs and time for optimizing assays can be avoided and reduced, respec tively, as well as reliability improved.
Literatur [1] Miller, J.J. (2004) Clinical Laboratory International 28, (2), 14-17 [2] Kusnezow, W., Hoheisel, J.D. (2003) J. Mol. Recognit. 16, 165-176 [3] Patton, W.F. (2000) Electrophoresis 21, 1123-1144 [4] MacBeath, G. (2002) Nat. Genet. 32, 526-532 [5] Miller, J.J., Valdes, R.Jr. (1992) J Clin Immunoassays 15, 97-107 [6] Wood, W.G. (1991) Scand. J. Clin. Lab. Invest. Suppl. 205, 105-112 [7] Kricka, L.J. (1999) Clinical Chemistry 45,(7), 942-956 [8] Span, P.N., Grebenchtchikov N., Geurts-Moespot, J., Sweep, C.G.J. (2003) Clinical Chemistry 49,(10), 1708-1709
Abb. 10 ELISA with lysates of a human kidney-cell carcinoma. Left side PBS/NaCl/Tween 20 and right side with novel CrossDown Buffer (AppliChem) Lane A-G serial dilution of immun sera; Lane H: preimmun sera; Column 5: blank. (Dr. Specht, Para Bioscience GmbH).
blocking
Without Blocking ... No Result!

This is a short message describing the necessity to block surfaces in immunoassays.
Dr. Wolfram H. Marx, AppliChem, Dr. Astrid Voigt, University Hospital Jena, Dr. Tronhung Quang, Dr. Rainer Klocke, Prof. Dr. Sigrid Nikol, University Hospital Mnster, Dr. Christoph Specht, PARA Bioscience GmbH
Many different blocking protocols exist and they work in various assays sometimes better, sometimes worse. However, universal blocking solutions do not exist.
All kinds of immunoassays require blocking to prevent nonspecific binding of antibodies or components of the sample to surfaces e.g. ELISA plates or Western blot membranes. Otherwise, this binding would lead to a strong background, falsify or even destroy results. Effi cient blocking means no areas on surfaces are available for nonspecific binding. That's the theory. In practice, the devil is in the details. A review of the literature shows that for every detection method using antibodies, several hundred blocking protocols exist describing variations of blocking solutions based on different blocking reagents. They all have in common that cer tain molecules are present in vast excess to cover the entire surface. Frequently used blocking reagents contain BSA, gelatin from fish, nonfat dried milk, casein or synthetic molecules. Unfortunately, the optimum reagent has to be determined for each new assay, since they all have certain restrictions when used with real samples such as blood, serum, cell lysates or tissue sections.
pose special problems since many analytes are coupled to BSA to achieve a better immunization. It is no won der that some antibodies will bind with high affinity to BSA. In this case, blocking would lead to an even increased background due to the reaction of antibodies with the blocking reagent. Highly purified Caseinbased reagents have pro ven to meet most of the important criteria of the ideal blocker but the preparation of such a reagent requires experience of a skilled person. Hydrolysis of casein must be performed over many hours and any mishand ling may lead to precipitation of casein.
Blocking Buffer I - ready-to-use

AppliChem now offers Blocking Buffer I which meets all criteria. It is based on highly purified, chemically modified casein with an optimum distribution in terms of fragment size. This ready-to-use buffered solution requires no assay optimization and is stabilized with ProClin 300 instead of toxic additives like thimerosal or sodium azide. Nevertheless, even effective blocking may not pre vent some background, caused by matrix effects or cross reactivity. Additionally applying AppliChem's CrossDown Buffer shall reduce or even abolish nega tive effects in different immunoassays such as immuno histochemical staining, Western blots or ELISAs.
Ideal Blocking
What properties are a must for the ideal blocking reagent? First, it has to completely cover the surface and this is achieved best, if it contains molecules of different sizes where large and small gaps will be covered simultaneously. Secondly, the blocking rea gent shall not react with or bind to any components of the sample or the antibodies. BSA blocking reagents
10
Just theory? This is the practice!
heart brain
Immunohistochemistry The antigen Nestin was detected by ABC-immunocytochemical staining with alkaline phosphatase on Cytospin preps of the neuroblastoma cell line SK-N-LO. While a standard blocking buffer based on 1 % BSA in PBS stained large parts of the whole surface (upper panel), using the ready-to-use Blocking Buffer I led to a reduction of background staining (lower panel). Correct staining of the cytoplasmic Nestin is now clearly separated from the hematoxylin staining of the nucleus.
heart brain
Western-Blot Detection of the antigen CaMK II in lysates of heart and brain tissue of C57Bl/6 mice by Western blotting. After separation by SDS-PAGE, p roteins were transferred to a nitrocellulose Optitran BA-S 83 membrane (Schleicher & Schuell) and detected by ECL. The primary antibody was a monoclonal anti-CaMK II antibody (BD Bioscience), and the secondary antibody a polyclonal HRP-conjugated antibody (Santa Cruz). Blocking with 5 % non-fat dried milk in TBS-Tween is shown on the left side. This Western blot cannot be interpreted. A combination of B locking Buffer I and CrossDown Buffer as antibody dilution buffer for the primary and secondray antibody (right side), made the identification of the bands possible.
with Standard Assay Buffer
with CrossDown
false-positive binding
false-positive binding ELISA This ELISA (developed by PARA Bioscience, Gronau, Germany) detects immunoglobulins from guinea pig and is used for immunotoxicological studies with guinea pigs. The false-positive binding observed in the c ontrol lane A1A12 and the blind values (lane H1H12) prevents i nterpretation of the assay.
Use of CrossDown Buffer abolishes false-positive binding and allows a concentration-dependent detection (lanes B to G16 and B to G712). The capture antibody Goat-anti-guinea pig-IgG F(ab')2 and detection antibody Goat-anti-guinea pig-IgG F(c) biotinylated were from Jackson ImmunoResearch Laboratories, Inc. (concentration range each 0.3110 g/ml in PBS). Guinea pig IgG was diluted either in CrossDown Buffer or PBS (columns 16 50 ng/ml; columns 712 10 ng/ml). PBS-BSA buffer was used as a blocking buffer and detection was c arried out with streptavidin-peroxidase (Sigma) and ortho-phenylenediamin (Sigma).
11
tips & tricks

Which substances may be added to antibody-containing solutions as preservatives?
Frequently, in biomedical laboratories sodium azide (final concentration 0.02-0.2 %) or Thimerosal (final concentration 0.005 %) are added to reagents. Both substances are toxic and less harmful alternatives are wanted. For such applications, ProClin 300 may serve as a substitute, if growth of bacteria and fungi/molds has to be prevented.
Is it possible to store antibodies in 50 % glycerol?
Does it make sense to add albumin?

Principally, it is good to add albumin, because albumin stabilizes antibodies. For long-term storage of antibodies in solution, we recommend the use of our Antibody Stabilizer (prod. no. A7148 and A7135), because they contain special ingredients stabilizing the structure.
If you really want to freeze antibodies, we recommend testing the performance of the antibody after thawing, because nearly every antibody cannot be frozen without losing part of its activity. In case the test is positive, prepare small aliquots and freeze. Dont re-freeze and thaw again, as the loss of acitivity will be greater with every freeze and thaw cycle. Be careful during thawing, as thawing too fast may damage your antibody in solution. A modern way of long-term storage of antibodies is storage in our reagents Antibody Stabilizer-PBS or -Tris. You may dilute the antibody in Antibody Stabilizer and directly store at 2-8C. Most antibodies show extremely long shelf lives, if stored in Antibody Stabilizer of around 5 years or more. One of the main advantages is that you dont have to aliquot for storage. For any new assay, you simply may take just the quantity of antibody you really need right now. The antibody is always ready-to-use for your next experiment as you dont have to slowly thaw.
Selection Guide
CrossDown Buffer Sample Buffer T+ Sample Buffer TBlocking Buffer I Blocking Buffer II EGrade Blocking Buffer III BSA
ELISA, EIA, RIA
Western Blot
Protein Array
Immunohistochemistry
Immuno-PCR
12
Washing Buffer TrisT+ Washing Buffer TrisTCoating Buffer Stripping Buffer I
12
Give it to me!
AppliCations
No.1
gy
gene technology demonstrate
Nucleic Acid Decontamination with The ExitusPlus Technolo

Advanced experiments in
that even small amounts of free DNA molecules are sufficient to cause infections, recombination or biological transformation [1,2]. The complete decontamination of equipment and surfaces from any DNA molecules is important for biological containment and safety, as well as preventing artifacts in PCR amplification experiments. Using new methods that detect extremely low levels of DNA molecules, we investigated the molecular mechanism of action of various commercially available DNA decontamination reagents. We found that when using high concentrations of DNA and short incubation times, none of the conventional reagents destroyed DNA molecules efficiently, despite their corrosive or even toxic properties.
AppliCations
Biological Buffers
AppliCations
No.4
kits from manufacturers is easy and convenient. Somethere is no kit available with kits such as their limits of good suppliers may be ELISA is required because characteristics of the available Ready-to-use ELISA kits from
AppliCations
No.6
Improving quality of ELISA

Using ready-to-use ELISA
Size-Exclusion Chromatograph y for purification of biomole cules
No.2
times however, home-made the right antibodies or the
detection are not appropriate. Many biochemical processes are markedly impaired by even small changes in the concentrations of free H ions. It is therefore usually necessary to stabilise the H concentration in vitro by adding a suitable buffer to the medium, without, however, affecting the functioning of the system under investigation. A buffer keeps the pH of a solution constant by taking up protons that are released during reactions, or by releasing protons when they are consumed by reactions. This handout summarizes the most commonly used buffer substances and respective physical and chemical properties.
completely different story.
Keywords Nucleic acid decontamination DNA degradation test Autoclaving DNA PCR test
All or Nothing at All Destruction and elimination of nucleic acids depends to this day, on bleach, a corrosive and toxic substance. To address issue, AppliChem partnered up with this multiBIND Cologne, to develop a new and unique nucleic acid decontamination technology: the ExitusPlus family of products, comprising DNA-ExitusPlus, and Autoclave-ExitusPlus. Comparing DNA-ExitusPlus to conventional products, we can demonstrate that it is fast and efficient in destroying nucleic acids without harmful or toxic effects on lab workers, equipment and the environment. Most decontamination reagents are based on several molecular principles for the destruction or inactivation of material: Modification and denaturation genetic can mask, but do not destroy the genetic information encoded in DNA strands and there is the risk that they may be chemically re-activated. Thus, safe and complete DNA decontamination depends on the degradation of DNA into very small fragments. Figures 1 and 2 show the results of comparing produced by the novel DNA-ExitusPlus the fragmentation process with conventional reagents. Using highly sensitive detection assays, it is seen that an efficient and total fragmentation was only obtained with DNA-ExitusPlus, whereas only partially degraded DNA pieces, some of which contained complete genetic information, were found in the decontamination fication or denaturation methodology. products that relied on modiSequence-Independent Degradation of DNA Only Applichems DNA-ExitusPlus is capable of achieving rapid and efficient degradation of nucleic acids, because unique method of action is based on chemical its and not enzymatic activity. Therefore, its effects on fragmentation are totally independent of the size and sequence of the DNA fragments. Larger plasmids require a longer incubation time than ones (e.g. primers). Assuming a theoretical smaller nicking activity of 100,000 nicks per minute, all DNA fragments will be destroyed, after several minutes, regardless of their size. Smaller fragments will disappear before the larger ones. Applying this theory to a test molecule (ccc form, 6 kb plasmid) only a small fraction of fragments with 200 to 500 bp in size will remain after 5 minutes. The nicks will be introduced statistically at any site, leaving not a single class of fragments. Remaining fragments are fully destroyed after 10 minutes.
because after storage of some
stored for two years at 4C without any problem. With home-made ELISA it is a For any new measurement one has to coat a new plate, days the plates dont perform as well as before.
Size-exclusion chromatography (SEC) is a popular method to separate biomolecules based on their size. Primarily, it is applied to the separation of biopolymers such as proteins and nucleic acids, i.e. water-soluble polymers. This system is also called gel filtration, typically with beads of dextran or agarose serving as gel matrix. Smaller molecules pass significantly slower through the column than larger molecules. Not to be mixed up with gel electrophoresis, there are big differences in terms of the separation principle. SEC does not require electric current and the sieving effect will not separate small molecules first.
AppliCations
Improving Separation During Electrophoresis
SeparateIT gels represent a novel gel matrix for DNA
No.7
Keywords Keywords Immunoassays Antibody Stabilisation ELISA Plates Gel filtration matrix Why is there such a great difference in storage between home-made ELISA and ELISA kits? The reason is that in professional ELISA kit production the plates are not only blocked after coating, but also stabilised. easy to perform process has been an industry This standard for thirty years. For stabilisation of a plate one has to incubate with a coating stabiliser solution. It is just as simple as a second blocking step. But there were no such high quality stabiliser lutions freely available in low volumes for souse in research lab until now. AppliChem now offers a product for use in every research in volumes starting as small as 50 ml, which lab is called the AppliCoat Plate Stabiliser (Cat. No. A7708). This stabiliser solution is easyto-use and has a great advantage compared to almost any stabiliser used in industry. It gives better storage stability for coated antibodies and antigens than most other products do. And there is a second benefit with this product: Two benefits with one solution When antibodies are coated onto ELISA plates, most of the antibodies are not active. When the antibodies (or any proteins) into close contact to the plastics surface come of the ELISA plate, conformational changes can occur due to surface-protein interactions. The result is that most antibodies coated on a plate are unfolded or inactive. Only around 28 % of all coated antibodies active and can bind to analytes and this remain is greatly variable depending on the surface characteristics of the ELISA plate, which really differ from batch to batch or even can from well to well. These differences from well to well can affect the variability of an assay, because the antibodies can be affected. If there way of refolding antibodies and of preserving was a antibodies from conformational changes during storage, this could help to decrease such variabilities in assay performance. This is a key benefit of AppliCoat Plate Stabiliser. It assists antibodies and coated proteins to refold and then to preserve active conformation over a long time. Thus it has two benefits: 1. Refolding antibody conformation of some of the of coated antibodies and 2. Preserving correct conformation during storage. These benefits are used for production of high-quality ELISA kits as well as in research applications Stabiliser the percentage of active antibodies now. Even with AppliCoat Plate will still be in the range of 28 %. But the great difference is that the variability from well to well and from plate to plate can be minimised in most assays by using AppliCoat Plate Stabiliser. Such effects depend on the used antibodies, but when ELISA are validated (e.g. according to Guidance for Industry: Bioanalytical Method Validation, FDA, 2001) or according to other validation strategies, the difference can be measured in many assays. The positive effects of AppliCoat Plate Stabiliser are shown in Fig. 1. A sandwich ELISA with a monoclonal antibody has been cross-linked dextran beads Desalting Buffer exchange Nucleic acid/ protein purification
Keywords chemical properties usefull pH range buffer preparation
Recommendations for the setting 1. Temperature
Practical Tips Preparing Buffer
Cross-reactivity
of the pH value of a buffer and
Solutions
Interfering effects storage conditions
Depending on the buffer substance, its pH may vary with temperature. It is therefore advisable, as far as possible, to set the pH at the working temperature to be used for the investigation. For instance the physiological pH value for most mammalian cells at 37C is between 7.0 and 7.5. The temperature dependence of a buffer system is expressed as d(pK )/dT, which describes the change of the pK at an increase of temperature by 1C. 2. Titration (i) Generally, the pH value is set using NaOH/KOH or HCl. Slow addition of a strong acid or base whilst stirring vigorously avoids local high concentrations of H or OH ions. If this is not done, the buffer substances may undergo chemical changes that inactivate them or modify them so that they have an inhibitory action (Ellis & Morrison 1982). (ii) Under stirring dissolves in the solution. Stir solutions CO gently for precise measurements of the pH value. (iii) If a buffer is available protonised form (acid) and the non-protonised in the form (base), the pH value can also be set by mixing the two substances. (iv) Setting of the ionic strength of a buffer solution (if necessary) should be done in the same way as the setting of the pH when selecting the electrolyte, since this value increases depending on the electrolyte used. (v) If other components are added the buffer (e.g. EDTA, DTT, Mg , b-Mercapto to ethanol) changes in the pH should also be considered and pH should be retested. (vi) In the presence of divalent metal ions carbonate or phosphate buffers may form precipitates . 3. How can microbial contamination of buffer solutions be prevented? (i) Sterilization by filtration through a 0.22 m filter unit or by autoclaving. (ii) Addition of 0.02 % (3 mM) sodium (iii) Storage at +4C. (iv) High-concen azide. tration stock solutions.
It is indeed correct that smaller molecules pass more slowly through the matrix than larger molecules. This is due to longer path the smaller molecules must the travel. The longer path arises from the pores of the beads. The smaller molecules enter the pores and go inside the beads. can Entering the pores creates a longer path for the smaller molecules. There is a relationship between the delay this causes and the molecular size. The larger molecules can not enter the pores and therefore have a shorter path. The larger molecules therefore travel faster through the column than smaller molecules. Advantages of SEC/Gel Filtration Some important features of the technique have to be mentioned. First, the separation of the ones is very effective and is performed under mild conditions, does mean biomolecules large molecules from the small volume of the eluate may be kept small keep their biological activity. The and therefore, the techniques is suited to concentrate samples. The Matrix AppliChems size-exclusion chromatography columns are packed with AppliXchange-G25 composed partially of polymerized dextran. M, a beaded composite material It exhibits high selectivity, high resolution purified with AppliXchange-G25 M are and chemical stability. Molecules separated according to size. The chemical interaction of gel matrix and molecules be separated is negligible. No absorption to takes place. Buffer and pH effects on resolution are minimal. The size exclusion cut-off for AppliXchange-G25 M is set at 10 kD for proteins and 10 bp for nucleic acids. Purified biomolecules significantly diluted when processed using are not AppliXchange-G25 M. The number 25 correlates to the water regain of the matrix. A grade 25 means that the dry matrix will take up 2.5 times its weight of water (1 g AppliXchange-G25 M absorbs 2.5 g water). This is a water regain of 2.5. Likewise, a grade of 50 gives a water regain of 5 grams (1 g AppliXchange-50 plus 5.0 g water.) The ability to absorb water is dependent on extent of cross-linking. Tighter cross-linking the limits the ability of the beads to swell and as a consequence the water regain lower. Less cross-linking gives a more is flexible bead and allows additional swelling. The extent of cross-linking has been optimized for a particular water regain. The water regain/extent of cross-linking affects the separation properties of the Larger water regain/less cross-linking gels. results in larger pores, which in turn retains larger molecules. Less water regain/more cross-linking results in smaller pores, which in turn retains only the smallest molecules. The separation efficiency is
electrophoresis. Gel polymers are arranged in a conceptually different way, in accordance with a new theoretical model of gel electrophoresis. SeparateIT gels selectively retard the migration of large molecules, so that DNA bands remain sharp but are more spread out relative to each other. Thanks to this increased spacing, resolving power of SeparateIT gels is at least twice higher compared to resolving power of any other gels, including polyacrylamide gels.
Keywords improved resolution Polyacrylamide Gel Electrophoresis SeparateIT Polymer Solution
The extraordinary resolving power of SeparateIT gels results from addition of a special polymer to a polymerizing containing a monomer and a cross-linker. solution While the gels with SeparateIT-like properties different polymers, chemical composition could be prepared with several and molecular weight of the polymer required a careful optimization for each particular monomer/cross-linker combination. In order to satisfy numerous requests from researchers who wish to improve resolving power of their acrylamide based AppliChem now provides SeparateIT gels, Polymer Solution. This polymer has been optimized for the gels with a acrylamide to N,N-methylene-bisacrylamid ratio of e of 29 : 1. The polymer is not optimized polyacrylamide gels. for denaturing, urea-containing SeparateIT Polymer Solution comes as a 10X solution. It should be mixed with a buffered solution of acrylamide and prior to addition of TEMED and ammonium Bis persulfate. Gels with SeparateIT Polymer Solution are prepared in the same way as any regular polyacrylamide gels. Likewise, the electrophoresis, gel staining and recording are carried out as usual. The increased resolving power of SeparateIT gels enables full separation of closely spaced bands on short gels. For example, a pair of fragments differing by 4 bp is usually resolved on less than 4 cm of gel length. Two DNA fragments in the 70 150 bp range that differ by 1 bp can be separated on SeparateIT gels that are 8 cm long. We recommend that Mini gel cassettes, which are 8 or 10 cm long, are used for casting acrylamide-Bis gels with SeparateIT Polymer Solution. Such relatively short gels will be previously required 20 30 cm long polyacrylamide appropriate for the majority of demanding separations which have gels. The use of shorter gels is beneficial easier gel preparation and handling, for several reasons, including lower cost of the gel materials, faster electrophoresis runs, and lower consumption gel staining reagents. In addition, a lower of amount of sample DNA needs to be loaded on a gel with a high resolving power compared to a gel with a low resolving power. This is the case because DNA bands that migrate a short distance sharper than the bands that migrate remain a long distance. It is always advantageous when closely spaced bands separate after migrating just a few centimetres.
AppliCations
Colorada: A New Generat Of Fluorescent Labels ion
The Colorada product line today. Dye structures were is a selection of the newest carefully chosen to provide
No.8
AppliCations
CheLuminate: Improved Chemiluminesce
AppliCations AppliCations
SafetyFirst Caps: Safety-Systems for HPLC pBARN Cloning Vectors Positive Selection of Recomb inants
The BARN PCR Blunt Cloning barnase gene expression Systems -1 and -2 are based by cloning of PCR fragments on the disruption of toxic into the multiple cloning site vector are killed In
No.11
fluorescent labels available
No.9
mances, improving bioanalytical assays to the highest level. Eight different basic structures with precise absorption and emission maxima were selected to cover the full working range of wavelengths, starting from 400 nm up to 850 nm. The Colorada structures are based on 4 different chemical classes: coumarin, dipyrromethene borondifluoride, acenaphthopyrrole and cyanine
top-notch brightness perfor-
nce
(MCS) of pBARN vectors. E.coli upon plating. Therefore, the contrast to other positive
cells that contain non-recombinant
AppliCations
No.12
Cell Proliferation Assay XTT
Cell proliferation assays are widely used in cell biology for the study of growth factors, cytokines or media components. They are also applied in the screening of cytotoxic agents and lymphocyte activation. In order to determine the number of viable cells Cell Proliferation Kit XTT employs 2,3-Bis-(2-methoxy -4-nitro5-sulfophenyl)-2H-tetrazolium-5-carb oxanilide salt (XTT). Only in living cells mitochondria are capable to reduce XTT to form an orange colored water soluble dye. Therefore, the concentration of the dye is proportional to the number of metabolically active cells. Keywords XTT assay cytotoxicity testing non radioactive assay quantitating and viability testing of cells The need for a reliable, sensitive and quantitative assay that would enable analysis of a large number of samples development of methods, such as incorporation led to the of radioactively labeled H-thymidine into DNA or the use of 5-Bromo-2deoxyuridine (BrdU) as a substitute for radioactive thymidine to label
Enhanced ChemiLuminescenc e Detection Kits for Horseradish Peroxidase in Western / Southern / Northern Blotting The peroxidase-catalyzed nm. The incorporation of a
No.10
blue/white screening is not
required for this system.
cloning systems BARN-1/-2
of special E.coli strains nor In summary, pBARN cloning
are not restricted to the use the ligation of inserts only. of positive cloning systems available.
to a few restriction sites for vectors combine advantages
oxidation of luminol produces so-called enhancer into the
Keywords high absorption coefficients high fluorescence quantum yields wavelengths from 400850 nm coumarin BDP acenaphthopyrrole cyanine
Coumarin dyes are chemically and photochemically near UV / blue spectral region. Dipyrrometheneboron robust, relatively small dyes with good absorption coefficients in the difluorides (BDPs) are chemically and pH insensitive. They have high absorption photochemically robust, and coefficients and excellent fluorescence quantum yields. Trimethine cyanine dyes have very high absorption coefficients, and are available in anionic (A), cationic 8-Oxo-8H-cyclopenta[a]acenaphthylene-7-carbonitriles (c) and zwitterionic (Z) form. (Acenaphthopyrrole dyes) are chemically robust, pH insensitive dyes with high and photochemically absorption coefficients and fluorescence quantum yields. Pentamethine cyanine have extremely high absorption coefficients, dyes good quantum yields of fluorescence and are available in anionic (A), cationic (C) and zwitterionic (Z) form. Heptamethine indocyanine dyes have extremely high absorption coefficients and good quantum yields of fluorescence in the near infrared (NIR) spectral region. They are available in anionic (A), and cationic The dyes with a Large Stokes Shift (Colorada (C). 670 LSS and Colorada 690 LSS) belong to the chemical class of heptamethine indocyanines as well. These dyes have high absorption coefficients in the far red and good quantum yields of fluorescence the near infrared (NIR) spectral region, in with a large stoke shift (> 100 nm). To meet the need of labeling the broadest range of target molecules each fluorescent compound is available with many different reactive groups (e.g. the commonly used amine reactive and thiol reactive groups) while retaining the photophysical features of the parent dye. The reactive groups include carboxylic acid (- COOH), active ester triazine, aliphatic amine (- NH ), azide (- N ), iodoacetamide and 2-pyridyldisulfideamine (- COOX, NHS ester), dichlorofor labeling -SH in peptides or proteins. (PDA), the latter being very useful Furthermore, a new level of chemistry has been developed to provide the same fluorescent label with different charge (anionic, cationic, zwitterionic and neutral). values It is thus easy to find the ideal product among the Colorada series starting from the desired absorption/emission peak maximum and subsequently decide both the reactivity and charge properties.
maric acid.
into a glow and greatly improves the analytical characteristics of the reaction in terms of increased signal intensity and duration [1,2]. Typical enhancer compounds are substituted phenols, the most popular being p-iodophenol and p-hydroxycou-
a weak flash of light at 425 buffer forces the flash signal
without the known disadvantages
of other cloning systems
Keywords Luminol Redox Mediator Enhancer Long light emission
The enhanced, HRP catalyzed oxidation of luminol is a complex, multi-step reaction. While enhancers are useful in improving enzyme turnover and increasing the equilibrium concentration of a key intermediate, the luminol radical anion, work [3] has shown that, by addition recent of a suitable catalyst, a further, large increase in light output is obtained:
In analytical laboratories, it is common practice to seal bottles of HPLC solvents simply with a perforated aluminium foil or plastic cap. Connecting tubes are inserted into the bottles through the holes in the foil/cap without any additional sealing. However such basic covers do not seal the solvent containers efficiently. On entering an HPLC laboratory, one can often already smell the solvent vapors. This uncontrolled evaporation of solvents is not only a potential health hazard but also the loss of solvent by evaporation interferes with the quality and reproducibility of HPLC runs. We present here quantitative data comparing the efficiency of various sealing methods, including Applichems SafetyFirstCaps for HPLC. Keywords health protection To test for the reproducibility of the retention time and the loss of solvent by evaporation, of 31 days. The separation of three PAHs tests were performed over a period (Polycyclic Aromatic Hydrocarbons) with solvents, stored in either tightly bottles (SafetyFirst Caps) or bottles closed closed with caps with holes of different diameters, was compared. The results of the tests were dramatic. Test Conditions
A This Bottle was closed with a SafetyFirst Cap, fitting precisely the standard GL45 glass bottle thread used. Bottle B This bottle was tightly closed with the GL45-cap provided including a Teflon-foil seal. Bottle C This bottle was closed with a cap having a 10-mm hole leaving an opening of an area of approximately 0.785 cm . Bottle D This bottle was closed with a cap having 3 holes, 3 mm each, leaving an opening of an area of approximately 0.212 cm . Bottle
Keywords Molecular Cloning Vector PCR Blunt Cloning Positive Selection Barnase
Introduction Many cloning vectors in use today employ a blue/white screening approach to identify successful fragment insertions. DNA fragments (such as PCR products or pieces of a genome) are inserted into a multiple cloning site (MCS) within DNA vector. Then, transformed competent a plasmid E.coli cells are grown in the presence of X-gal. If the insertion was successful, bacterial colony will be white; if not, the the colony will appear in blue color. This traditional method, developed in seventies, has since been honed and the late simplified. However, it still has two major drawbacks: low efficiency of blue/white selection and high costs per cloning reaction. In addition, false-positive clones are frequently detected, making necessary sub-cloning and further time consuming investigation of candidate colonies, i.e. performing analytical restriction enzyme digest or sequencing. To facilitate more simple and efficient high-throughput cloning several positive selection cloning systems have been developed (Table 1.). The principle of positive selection cloning is rather straightforward. the MCS of a positive selection vector Ligation of a PCR fragment into disrupts the expression/activity of the toxic gene, permitting growth of positive binants (up to 100 %) upon transformation recomonly. E.coli cells that contain non-recombinant Disadvantages of known cloning systems vectors are killed upon plating. that also employ positive selection of recombinants poly-linker (MCS) in the middle of the include: (i) The position of the toxic gene. This results in limitations regarding the choice of restriction enzymes well as limitations of using universal as sequencing primers. (ii) Limitations for the bacterial strains to be used. (iii) size of the DNA inserts. (iv) In some cases Limited handling is cumbersome. The pBARN PCR Blunt Cloning System is an easy-to-use, fast and highly efficient positive cloning system of blunt or blunted PCR fragments. Fragments generated by either Taq DNA polymerases or other DNA polymerases without proof-reading activity require a filling-in reaction for optimal cloning efficiency. The pBARN PCR Blunt Cloning System is based on the toxic barnase gene product, a small, highly active ribonuclease from Bacillus amyloliquefaciens (Yazynin et al. 1996; Yazynin et al., 1999; Fig. 1). pBARN-1 and -2 PCR Blunt Cloning Vectors (3.4 kb and 3.2 kb, respectively), the main component of these systems, allow direct selection of recombinants via disruption of the lethal barnase gene, once expressed. The vector contains the barnase gene under the control of the LacZ promoter, N-terminally fused to an especially designed sequence, including the multiple
As a result of this breakthrough, the family of AppliChems CheLuminate-HRP chemiluminescent substrates has been developed, with specific formulations to meet the various requirements for immunoblotting optimal substrate for your application). (see table below for selecting the Outstanding Sensitivity The intense light output generated by the CheLuminate-HRP corresponding improvement in sensitivity. substrates translates into a Even the economical CheLuminate-HRP PicoDetect Extended formulation provides a sensitivity level at least eight times higher than with phenolic enhancers, down to the Detection limits are lowered further with CheLuminate-HRP FemtoDetect, to mid-femtogramlow picogram (10 ) range. level in sensitivity, is achieved with CheLuminate-HRP (10 ), while the ultimate FemtoDetect Plus, which is designed to detection. provide femtogram (10 )-level Long Signal Duration All substrates exhibit long light emission. However, this feature has been specifically maximized in the formulation developed for the CheLuminate-HRP FemtoDetect substrate, which offers a 24-hour light emission least ten times longer than with standard at phenolic enhancer substrates.
reduction of contaminant concentrations constant HPLC-retention time save solvents
DNA in living cells. The above methods have a number of disadvantages, including: use of radioactive materials and relatively techniques. The use of tetrazolium salts, complex such as MTT, commenced in the 1950s, is based on the fact that living cells reduce tetrazolium salts into colored formazan compounds.
Procedure Atthestartofthetest,all4bottleswerefilledwiththesame mixtureofWater+Methanol=20+80(w/w). UsingBottleBasareference,chromatogramsofamixtureof thethreePAHs(Naphthalene,Pyrene,Chrysene),werecom with the reference. pared Afterthefirstmeasurement,allbottleswerekeptatroom temperatureunderafumehoodwithagentleairflowfor 31days. HPLC-System HITACHI LaChrom Elite system with Diode Array Detector under control of the EZChrom Elite Isocratic pump conditions with premixed Software. mobile phase. RP-18e (5), 125 x 4 mm
HPLC-Column Purospher
AppliChem provides background information or published application notes for some selected products.
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13
blocking
Comparing Blocking Reagents

Quality and economy now combined in a novel blocker
Dr. Wolfram H. Marx, AppliChem Blocking in immunoassays is an important step, i.e. the saturation of free binding sites on surfaces of ELISA wells or Western Blot membranes. Noncomplete blocking will be cursed with high background and either useless or nonmeaningful results. Therefore, several very different methods for blocking were developed and became established especially proteinbased blockers containing casein or BSA. The caseinbased blockers show an unsurpassing efficiency, but only when they are prepared using casein which has undergone severalfold cleavage. Whenever reproducible results with a low standard deviation are of importance, no serious alternative to casein exists. Unfortunately, preparation is complex making this product more expensive. Now, we present a new alternative, Blocking Buffer II EGrade, combining quality and economy.
State of the art

If samples are rare, expensive, or the results have to be reliable, then there exists no alternative to the highly purified caseinbased blocking reagents (e.g. Applichem's Blocking Buffer I). The only option avai lable is based on ultrapure BSA and in many test systems, efficiency is comparable. Differences appear in terms of standard deviations when real samples are measured (determined as coefficient of variation (CV) for multiple measurements). These differences are mainly due to differences in molecular size of the blocking agent. The relatively large BSA leaves gaps on the sup port surface which will be covered by smaller molecu les coming from the sample. Most of these substances do not influence the results. Some do interact with e.g. detection antibodies or the label such as peroxidase, phosphatase or fluorescent dyes. As soon as inter actions occur, they lead to erroneous results. The diffi culty is that one cannot predict when it will happen, how strong the interference will be and whether devia tions will shift results up or down. You can identify such interferences simply by observing high standard deviations, i.e. high CVs. Looking at the CV as shown in fig. 1, the problem is obvious. Figure 1 shows a sandwich ELISA, which seemed to work well by inclu ding a BSAbased blocking reagent (Blocking Buffer III BSA). But CVs tell something different. Upon first glance, the assay looks good but the results are unre liable. Not until running the same assay using
AppliChems Blocking Buffer I, is a reliable result achie ved. At first view, what appears as an unimposing effect, ultimately influences the significance of all subsequent tests. In most cases, ELISAs, Western Blots or other immunoassays simply serve as tools to verify or falsify a hypothesis of the whole research project. Those who use unreliable tools, as shown for the BSAblocked
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ELISA, must be prepared that their results will be ques tionable and, in the present case, doubts are justified. There is another problem arising from time to time that must not be overlooked. Over the years it has been common practice to couple small antigenes, socalled haptens, to BSA as carrier molecules to generate anti bodies. It works well, is reliable and simple. The pro blem is that a large number of commercially available antibodies as well as antibodies generated by and exchanged between scientists not only bind the hapten with significant affinity but also BSA. This wouldn't be a problem if the producers of such antibodies would mention the method of immunization. In those cases, one would be aware of the potential interference and would not choose a BSAbased blocking reagent. Unfortunately, the exact opposite is reality and research antibodies do not include this essential information. Ne vertheless, in most cases BSA is a very good blocking reagent. It is cheaper than optimized caseinbased blocker, even if the BSA" purification is laborious. The big disadvantages of good proteinbased blockers is the high costs and the search for alternatives has resulted in several varieties. Gelatin from fish or nonfat dried milk are just two to mention. In some laboratories and assays they meet the requirements and, in many other assays they constantly fail. Due to the uncontrollable quality of these raw materials, these blocking reagents are not used in medical research, e.g. in pharma assays, preclinical or clinical studies or in diagnostics, since reproducibility is not given. Repro ducibility of an assay depends on constant quality of the raw materials.
Alternatives wanted
Since the desired alternative needs to be of high, repro ducible quality but at low cost, synthetic blockers were investigated and Tween represents the first, best known and simplest solution. Other synthetic blockers are also availavle at low prices as well. While consistency of raw materials may be optimal, one problem couldn't be solved: reproducibility of results thereby limiting their use. Considering that the total costs of blocking are just a small part of the total costs of an assay, it is obvious why very few scientists would trade quality for marginal savings. Now, AppliChem presents THE new alternative for efficient and costeffective blocking in immunoassays. Blocking Buffer II EGrade is manufactured to DIN ISO 9001:2000 quality requirements, free of any serum pro teins, and peptidebased. Since the synthesis of the peptides is controlled, blocking is uniform and repro ducible from batch to batch. The costs are comparably lower than for proteinbased blockers, while quality is acceptable in contrast to synthetic blocker. 2010 AppliChem Immunoassay Buffer
coefficient of variation [%]
Blocking Buffer I
Blocking Buffer III BSA
Fig. 1 Comparison of the coefficient of variation (CV) of an ELISA with blood samples. The ELISA was performed once with Blocking Buffer I and once with Blocking Buffer III BSA under identical conditions. To determine the CV a complete ELISA plate was measured (n=96).
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OD 450 [nm]
Blocking Buffer I
Blocking Buffer II Egrade
synthetic Blocker
Fig. 2a OD-values for blocking over night with different blocking reagents. This is a modified analyte-free assay originally developed by Steinitz & Baraz (2000), measuring the influence of blocking without negative effects of real samples.
K-
BB I
BB III BB II
SyB
Fig. 2b Image of the ELISA plate described in fig. 2a. The left column is the control without blocking (K-). The order of blocking reagents is as in fig. 2a Blocking Buffer I (BB I), Blocking Buffer III BSA (BB III), Blocking Buffer II EGrade (BB II) and synthetic blocker (SyB).
CV [%]
Blocking Buffer I
Blocking Buffer II Egrade
synthetic Blocker
Fig. 3 Graph of the coefficient of variation (CV) of the assay from fig. 2. The lower the CV, the more reliable the assay. In industrial practice, CVs of < 15 % for ELISAs with real samples are needed (e.g. according to FDA guidelines). Please note that the CVs shown here only represent effects of blocking reagents, since the assay don't includes analytes and other negative effects.
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Analyte-independent comparison
Is it possible to determine the real blocking efficiency and quality of a new blocking reagent? According to the methods of Steinitz & Baraz (2000) and Steinitz (2000), a test system was established with the following changes: peroxidase was used instead of phosphatase as reporter enzyme, eight determinations instead of double values, and additional determination of the coefficient of variation (CV). The test included Blocking Buffer I, highly purified and multiply digested casein, Blocking Buffer III BSA based on BSA, the novel Blocking Buffer II EGrade, as well as a commercial, synthetic blocking reagent. The surface of the ELISA wells were blocked by the different blocking reagents and incubated with a detec tion antibody (analog to Steinitz (2000), but using peroxidase) without any additional additives. After washing, the staining reaction was carried out. The lowerthe optical density (OD), the better the blocking efficiency. Figure 2 shows blocking overnight. All variants do block. The blocking efficiency of the proteinbased blocking reagent is superior, good for Blocking Buffer II EGrade and also good for the synthetic product. The graph in fig. 2a shows the ODs, fig. 2b the correspon ding image of the blocked ELISA plate. The left column served as an unblocked control. The coefficient of variation of the same experiment is presented in fig. 3. Here, the wheat separates from the chaff. At first view, the synthetic solution looks pro mising. Even though a simple, artificial test system without any interfering substances was used, the CV value of 25 % is extremely high. Such a CV is transferred to the total CV of an assay according to the error propa gation of each separate error of the assay. It is question able whether a reproducible assay may be established with such a synthetic blocking reagent. The advantage of the novel Blocking Buffer II EGrade is clear. Blocking is highly reproducible with a constant CV, identical to the proteinbased reagents. Combining the results shown in fig. 2 and 3 allows one to draw the conclusion that in those assays, where Blocking Buffer II EGrade gives a low background, reproducabillity is seen also. This is an advantage over other alternatives like the fish gelatin, milk powder or synthetic blocking reagents. Blocking Buffer II EGrade combines economy and reproducibility for various assays, and may be regarded as a suitable alternative to the proteinbased blocking reagents. The comparison according to Steinitz also proves the unique position of proteinbased blockers for sensitive applications like pharma assays, clinical studies or other assays inclu ding valuable samples.
Conclusion
For many assays in LifeSciences, AppliChem offers an alternative to high cost proteinbased blocking reagents. Blocking Buffer II EGrade has the potential to combine economy and quality.
Literatur [1] Steinitz, M. & Baraz, L. (2000) J. Immunol. Methods 238, 143-150. A rapid method for estimating the binding of ligands to ELISA microwells. [2] Steinitz, M. (2000) Anal. Biochem. 282, 232-238. Quantitation of the Blocking Effect of Tween 20 and Bovine Serum Albumin in ELISA Mircrowells.
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Improving quality & stability of ELISA

Using ready-to-use ELISA kits from manufacturers is easy and convenient. Sometimes however, home-made ELISA are required, because there is no kit available with the right antibodies or characteristics, such as limits of detection are not appropriate. Ready-to-use ELISA kits from good suppliers may be stored for two years at 4C without any problem. With homemade ELISA kits it is a completely different story. For any new measurement one has to coat a new plate, because after storage of some days the plates dont perform as well as before. Why is there such a great difference in the suitability for storage between homemade ELISA and commercial ELISA kits?
The reason is that in commercial ELISA kit production the plates are not only blocked after coating, but also stabilised. This easy to perform process has been an industry standard for thirty years. For stabilisation of a plate one has to incubate with a coating stabiliser solution. It is just as simple as a second blocking step. But there were no such high quality stabiliser solutions freely available in low volumes for use in research lab until now. AppliChem now offers a product for use in every research lab in volumes starting as small as 50 ml, which is called the AppliCoat Plate Stabiliser (Cat. No. A7708). This stabiliser solution is easy-to-use and has a great advantage compared to almost any stabiliser used in industry. It gives better storage stability for coated antibodies and antigens than most other products do. And there is a second benefit. coated antibodies and 2. Preserving correct conformation during storage. These benefits are used for production of high-quality ELISA kits as well as in research applications now. Even with AppliCoat Plate Stabiliser the percentage of active antibodies will still be in the range of 2-8 %. But the great difference is that the variability from well to well and from plate to plate can be minimised in most assays by using AppliCoat Plate Stabiliser. Such effects depend on the used antibodies, but when ELISA are validated (e.g. according to Guidance for Industry: Bioanalytical Method Validation, FDA, 2001) or according to other validation strategies, the difference can be measured in many assays. The positive effects of AppliCoat Plate Stabiliser are shown in Fig. 1. A sandwich ELISA with a monoclonal antibody has been done. This monoclonal AB lost its binding activity when coated on a plate and stabilised only with BSA without applying AppliCoat Plate Stabiliser. Figure 1 shows a high temperature stress test at 37C for 84 days after application of the stabiliser solution. The real OD signal is shown without any normalisation, which could potentially falsify interpretation of the results. One can clearly see the better binding activity when AppliCoat Plate Stabiliser is used. If this plate was stored dry at 4C the test would correlate to around 2 years of storage.
An improved workflow for most labs

This advantage of a prolonged storage time can help to set up a completely new workflow in research labs. Whilst nowadays it is best practise to produce a new and fresh ELISA plate whenever some measurements with a homemade ELISA are done, this can change. One can easily coat, block and stabilise as much plates as needed for the next 3 to 6 months. Those plates are dried in an incubator at 20C or 30C for 60 to 120 minutes, the plates can be stored in the fridge for several months. Those plates can be used from time to time for measurements. All those plates are therefore produced in one batch in your lab minimising plate-to-plate variations coming from differences in plate production. Thus the new workflow in labs has three great advantages: 1. Less work, because coating and blocking of many plates can be done in one run instead of doing this work for any new plate again and again. 2. Lower variations in measurements from plate to plate as described above. 3. Saving time and potentially improving the quality of the results of a lab.
Two benefits with one solution

When antibodies are coated onto ELISA plates, most of the antibodies are not active. When the antibodies (or any proteins) come into close contact to the plastics surface of the ELISA plate, conformational changes can occur due to surface-protein interactions. The result is that most antibodies coated on a plate are unfolded or inactive. Only around 28 % of all coated antibodies remain active and can bind to analytes and this is greatly variable depending on the surface characteristics of the ELISA plate, which can really differ from batch to batch or even from well to well. These differences from well to well can affect the variability of an assay, because the antibodies can be affected. If there was a way of refolding antibodies and of preserving antibodies from conformational changes during storage, this could help to decrease such variabilities in assay performance. This is a key benefit of AppliCoat Plate Stabiliser. It assists antibodies and coated proteins to refold and then to preserve active conformation over a long time. Thus it has two benefits: 1. Refolding of antibody conformation of some of the
Industrial technology now available even for small research labs

According to the needs of research labs AppliCoat Plate Stabiliser (A7708) is available in volumes of 50 ml, 125 ml and 500 ml. It can be used in combination with standard blocking solutions like casein (e.g. Blocking Buffer I, A7099) or BSA based solution (e.g. Blocking Buffer III BSA, A7252) and other blockers. For any researcher this industrial stabilisation technology is made available by AppliChem. Even small labs can make use of it. Test it for yourself!
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Figure 1. Coating, blocking and stabilization of ELISA plates in a 3 step and in a 2 step process. The solutions are added, incubated and removed again. Then the plates are dried and sealed in plastic or aluminium foil for dry long-term storage
Figure 2. Stress test at 45C with a monoclonal antibody. This antibody showed even after stabilization with carbohydrate containing solutions of the first generation no binding functionality after 24 hours (data not shown). The stabilizers of the second generation A, B and C show a cceptable stabilization. After stabilization with AppliCoat Plate Stabilizer, a modern stabilizer of the third generation, this unstable antibody shows even after 66 days of incubation at 45C more than 85% of its activity and therefore an impressively enhanced stability.
Figure 3. Calibration curves of an ELISA are shown, which was either blocked only with BSA (red) or blocked with BSA and additionally stabilized with AppliCoat Plate Stabilizer (blue). Calibration curves at the starting day are identical. The sensitivity of the assay without stabilization d ecreases rapidly during storage at 37C. During this stress test the ELISA after stabilization does not show a decrease in sensitivity even after 84 days of incubation.
Figure 4. Binding activity of an ELISA at the starting day and after storage for 84 days at 37C is shown. In fig. 3 the complete calibration curves are shown (only for BSA blocking). Here we show the measurements at 40 ng/ml. Activation of binding capacity by AppliCoat Plate Stabilizer can be seen quite clearly. It is also shown, that AppliCoat Plate Stabilizer can be combined with different blockers without any problems.
How to use AppliCoat Plate Stabiliser?

Figure 1. shows the simple three step production process, which can be used for kit production as well as for homemade ELISA. 1. Coating as routinely. 2. Blocking as routinely. 3. lates are washed 3 times with 200-300 l PBS or a ashing Buffer without P W detergents (e.g. Washing Buffer TrisT- (10x) order number A7137) 4. dd 200 l AppliCoat Plate Stabiliser and incubate for 30-90 minutes at apA prox. 20-30C 5. emove AppliCoat Plate Stabiliser by suction. Incubate the plates at 37C R until dry. This needs around 60 to 120 minutes depending on incubator type, number of plates in the incubator and speed of air circulation in the incubator. 6. tore the plate with a lid in the fridge for 3 to 6 months. Alternatively plates can S be sealed with plastic or aluminium foil under dryness. When plates are stored dry, the shelf life will be around 1 to 3 years. Achievable shelf life can differ from antibody to antibody. The used plates, coating concentration, procedures and buffers have additional impact on achievable stability. Thus if plates should be stored for more than 6 months, a stress test at 37C or 45C for storage should be done with the plates to easily measure the shelf life of the plate in a short time.
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less is more!
Sometimes,
For The Sake Of The Environment

Many reagents prepared in biomedical research labs are ideal nutrient broths for unwanted germs (bacteria, fungi). To prevent their growth, reagents are either autoclaved, sterile filtered, or antibiotics / antimycotics or toxic substances are added. One of these additives is Thimerosal, a mercury-containing molecule which is dangerous for the environment. Our original immunoasssay buffer contained this chemical too, but now we have replaced it by the nontoxic ProClin 300. For the sake of the environment.
For Better Results For Your Safety

We would like to keep you healthy so that you stay our customer. FYI: Thimerosal is classified as toxic. The lethal dose (rat, s.c.) is 9 mg/kg, compared to ethidium bromide with a lethal dose (mouse, s.c.) of 110 mg/kg. In some countries, Thimerosal is a forbidden additive. Changing the composition has no negative influence on the performance of the products. With CrossDown and all other immunoassay buffers you are even in a better mood and your immunoassays show a better quality.
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products
Instructions for use
Antibody Stabilizer-PBS
Stabilization buffer for long-term storage of antibodies and proteins
A7148
A7148,0050 A7148,0125 50 ml 125 ml
pH value pH 7.4 0.2 Preservative contains 0.1 % ProClin300 Storage 2-8C. Avoid freezing * Stability at 2-8C: 2 years * Upon freezing, an insoluble precipitate is formed. The activity is not significantly reduced!
Antibody Stabilizer-PBS is made for long-term storage of proteins and antibodies at 2-8C in solution. Components of Antibody Stabilizer conserve the structure of proteins and antibodies in solution. Thus proteins and antibodies are prevented from loosing functionality during storage. Usually, antibodies / proteins are diltued in Antibody Stabilizer-PBS at least by a factor of 1:20, but significant further dilution is possible. The typical concentration of antibodies during storage is 400 to 1000 ng/ml. Many antibodies may be stored even at concentrations as low as e.g. 80 ng/ml in Antibody Stabilizer-PBS, without significant loss of binding activity within the first two years. Such low concentrations do not require pre-dilutions before every single application. Immediately before use Antibody Stabilizer-PBS should be mixed thoroughly. Just dissolve proteins or antibodies with Antibody Stabilizer-PBS for storage in the refrigerator. You can also use Antibody Stabilizer-PBS for storage of coated ELISA plates. After blocking add Antibody Stabilizer-PBS to the coated plates. In this manner treated plates can be stored for a longer time in the refrigerator. Stability of proteins and antibodies differs significantly from case to case. Stability depends on characteristics and concentration of the used proteins and antibodies. The user has to test it therefore with its own proteins or antibodies. Antibody Stabilizer-PBS is ready-to-use.
Antibody Stabilizer-Tris
Stabilization buffer for long-term storage of antibodies and proteins
A7135
A7135,0050 A7135,0125 50 ml 125 ml
pH value Preservative Storage Stability
pH 7.2 0.2 contains 0.1 % ProClin300 2-8C. Avoid freezing * at 2-8C: 2 years

Antibody Stabilizer-Tris is made for long-term storage of proteins and antibodies at 2-8C in solution. Components of Antibody Stabilizer-Tris conserve the structure of proteins and antibodies. Thus proteins and antibodies are prevented from loosing functionality during storage. Usually, antibodies / proteins are diltued in Antibody Stabilizer-Tris at least by a factor of 1:20, but significant further dilution is possible. The typical concentration of antibodies during storage is 400 to 1000 ng/ml. Many antibodies may be stored even at concentrations as low as e.g. 80 ng/ml in Antibody Stabilizer-Tris, without significant loss of binding activity within the first two years. Such low concentrations do not require pre-dilutions before every single application. Immediately before use Antibody Stabilizer-Tris should be mixed thoroughly. Just dissolve proteins or antibodies with Antibody Stabilizer-Tris for storage in the refrigerator. You can also use Antibody Stabilizer-Tris for storage of coated ELISA plates. After blocking add Antibody Stabilizer to the coated plates. In this manner treated plates can be stored for a longer time in the refrigerator. Stability of proteins and antibodies differs significantly from case to case. Stability depends on characteristics and concentration of the used proteins and antibodies. The user has to test it therefore with its own proteins or antibodies. Antibody Stabilizer-Tris is ready-to-use. ProClin is a registered trade mark of Rohm and Haas Company. 2010 AppliChem Immunoassay Buffer
21
products
AppliCoat Plate Stabilizer

Preservative for long-term storage of coated surfaces in immunoassays
A7708
A7708,0050 50 ml A7708,0125 125 ml A7708,0500 500 ml
pH 6.5 0.2 contains 0.1 % ProClin300 2-8C. at 2-8C: 1 year
Improving quality of ELISA measurements and enabling easier workflow in research labs
AppliCoat Plate Stabilizer is a ready-to-use reagent for the preservation of immobilized antibodies and proteins. AppliCoat Plate Stabilizer is simply added to the coated microtiter plates, polystyrene beads or glass slides. AppliCoat Plate Stabilizer seals by forming a uniform stabilizing layer over antibodies and antigens immobilized on the solid phase. This layer is distinguished by good solubility without affecting assays performed at a later date. It is used for long-term storage of precoated plates and other surfaces used in immunoassays. AppliCoat Plate Stabilizer is used directly after blocking and washing. AppliCoat Plate Stabilizer seals and stabilizes coated proteins and antibodies. In the case of strong background problems we recommend the use of AppliChem Blocking Buffer I (A7099) before sealing. Blocking Buffer I is characterized by a higher blocking efficiency than most other known blocking reagents. After incubation of the microtiter plate or solid phase with AppliCoat Plate Stabilizer, the plate can be stored either directly under moist conditions or after drying of the plate or solid phase. The shelf life of the coated molecules is substantially extended to typically 1 to 3 years when stored cool and dry. The assay buffer or the specimen can be added directly onto the sealed plate (solid phase) for use in the assay. An additional washing step is not necessary. AppliCoat Plate Stabilizer is free of proteins. The values for shelf life of sealed plates are to be used only as a guide. Longer shelf lives have been observed, but this may not be generalized for all assays. Therefore any assay has to be tested for its individual shelf life.

1. Apply commonly used coating and blocking procedure for microtiter plate. 2. After blocking: Wash 3 times with 200-300 l PBS or Washing buffer without detergents (e.g. AppliChem A7137). 3. Add 200 l AppliCoat Plate Stabilizer per well and incubate for 15-90 minutes at 20-30C. 4a. Moist storage: Cover the plate with adhesive film and store at 2-8C for up to 3 months. or 4b. Dry storage: Remove the AppliCoat Plate Stabilizer by aspiration. Incubate the plates at 37C until dry. Incubation typically takes 60 to 120 minutes, depending on temperature, incubator type, number of lates and the (active) air circulation in the incubator. Store the plates sealed in plastic foil or aluminium foil under dry conditions (where required with desiccant) at 2-8C for 1 to 3 years.
Figure 1: High temperature stress test at 37C for 84 days after application of the plate stabilizer solution. The true OD signal is shown without any normalisation, which could potentially falsify interpretation of the results. One can clearly see the better binding activity when AppliCoat Plate Stabilizer is used. When this plate would have been stored not at 37C but dry at 4C the test would correlate to around 2 years of storage Figure 2: Application of AppliCoat Plate Stabilizer. Stabilization is similar to a second blocking step. After this the ELISA plates are dried in an incubator and can be stored in a fridge for a long time.
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Blocking Buffer I
Solution for blocking unspecific binding sites for ELISA, EIA & Western Blots
A7099
A7099,0050 50 ml A7099,0125 125 ml A7099,0500 500 ml
Composition pH value Preservative Storage Stability
low-molecular weight highly purified casein with NaCl and Tween pH 7.2 0.2 contains 0.1 % ProClin300 20C or at 2-8 C at 20C: 1 year, repeated freeze / thaw cycles are possible at 2-8C: 6 months

Blocking Buffer I saturates free binding capacities on plastic consumables and other surfaces like ELISA plates and blotting membranes. Thus a reduction of unspecific binding on surfaces can be achieved. Efficiency of blocking is significantly improved in comparison to standard blocking procedures by a special production method, which leads to casein molecules with many different molecular sizes. Blocking Buffer I can be used in ELISA, EIA, RIA, Western blotting, immuno-PCR, protein arrays as well as immunohistochemistry. Immediately before use the buffer should be mixed thoroughly. Blocking Buffer I is ready-to-use. Repeated freezing and thawing is possible. After immobilisation of capture antibody or target protein Blocking Buffer I is applied without dilution in wells or on membranes. Incubation time has to be adopted depending on surface characteristics by the user. We recommend blocking over night at 4 C, but in many cases shorter incubation is also promising. After blocking the surface has to be washed with Washing Buffer to make it useable for the next working steps.
Blocking Buffer I Background reduction Extremely efficient background reduction even in critical assays For use in all immunoassays
Other blockers Commonly known background reduction Some products only for use in ELISA or Western blotting, many different specialised products For some products pre-dilution with other buffers recommended Usability depends on product, negative quenching with fluorescent dyes has to be checked with some products Normal effects on validation, no decrease in variations shown Cooling or freezing for long-time storage, cooled transportation for many products recommended
Usability
Ease of application Usability with different detection methods Effects on validation (e.g. for new FDA-guidance for industry) Storage and transportation
Ready-to-use Usable with all common detection methods very good results with peroxidases, phosphatases and fluorescent labels Positive effects, variations decrease, background effects are avoide, validation criteria are met easily Cooling and freezing for long-time storage, repeated freezing and thawing possible, but no cooled transportation needed
23
products
Blocking Buffer II EGrade
A7516
A7516,0125 125 ml A7516,0500 500 ml
Solution for blocking unspecific binding sites for ELISA, EIA, Western Blots, Protein Arrays, Immuno-PCR peptide-based blocking buffer; free of serum and BSA, phosphate-free pH 7.0 0.2 contains 0.1% ProClin 300 20C or 2-8C Immediately before use the buffer should be mixed thoroughly at -20C: 1 year, repeated freeze / thaw cycles are possible at 2-8 C: 6 months
Blocking Buffer II EGrade is the most economic solution (Economical Grade) for blocking of unspecific binding sites. It is THE cost-effective alternative to the casein-based Blocking Solution I. effective blocking simple and economically BSA-free

In many assays, Blocking Buffer II EGrade prevents nonspecific and unwanted binding to surfaces. Blocking Buffer II EGrade saturates free binding sites on the surfaces of e.g. microtiter plates (plastic consumables), Western blotting membranes or slides, avoiding undesired binding of analytes or detection antibodies to surfaces. This leads to significantly reduced background and improved sensitivity of the assay. Suitable for many assays, Blocking Buffer II EGrade represents an excellent alternative to BSA-based blocking solutions. Problems with interactions and cross-reactivities arising from BSA, which are present in many blocking solutions, are avoided by the use of Blocking Buffer II EGrade , as it is free of serum proteins such as BSA. In case satisfying results cannot be obtained with the Blocking Buffer II EGrade , e.g. if your assay measures analytes in plasma, serum or tissue specimen, we strongly recommend using our Casein-based product Blocking Solution I instead (A7099). Blocking Buffer II EGrade is a ready-to-use reagent and applied like common blockers. Use Blocking Buffer II EGrade undiluted for incubation of surfaces to saturated free binding sites in non-problematic assays. Background and nonspecific binding can not only occur at surfaces, but also between antibodies and components of complex specimen. In this cases only an assay buffer, which is used for the immunological detection reaction, can lead to satisfactory results. Therefore we recommend using CrossDown Buffer (order no. A6485). CrossDown Buffer acts as a filter for binding, which doesnt affect high affinity binding in any way, but depletes nonspecific binding and interference like matrix effects and cross reactivities.
ProClin is a registered trade mark of Rohm and Haas Company.
Saturation / Blocking
1. If the plate was treated with reagents containing detergents please wash the plate 3 times in a wash buffer free of detergents (e.g. Washing Buffer TrisT-, Prod. No. A7137). If you have only used Coating Buffer (Prod. No. A7136 or A7150) aspirate Coating Buffer or empty plates by wrapping firmly onto paper cloth. 2. Add 200-300 l Blocking Buffer II EGrade to each well. Incubate at room temperature for 1-4 hours or overnight (mostly one hour is quite enough). Duration of blocking can be minimised by shaking the plate at 600-900 rpm. Duration of blocking depends on characteristics of used surface and has to be tested individually. 3. Aspirate Blocking Buffer II EGrade or empty plates by wrapping firmly onto paper cloth. Wash 3 times in wash buffer containing a non-ionic detergent, e.g Washing Buffer TrisT+ (Prod. No. A7158). Immunoassay Buffer AppliChem 2010 1. If the membrane was treated with reagents containing detergent please wash the membrane 3 times in a wash buffer free of detergents (e.g. Washing Buffer TrisT-, Prod. No. A7137). 2. Incubate membrane in Blocking Buffer II EGrade at room temperature for 1-4 hours or overnight (in most cases one hour is enough). The time for blocking depends on the characteristics of the membrane used and has to be tested individually. 3. a) Wash membrane 3 times in a wash buffer containing a nonionic detergent, e.g Washing Buffer TrisT+ (Art.-Nr. 7158). or b) add antibody for detection and continue incubation and detection.
microtiter plates
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membranes
24

Solution for blocking unspecific binding sites for ELISA, EIA, Immuno-PCR, immunohistochemistry & Western Blots Composition pH value Preservative Storage Stability Standard Blocking reagent with BSA and Tween pH 7.4 0.2 contains 0.1 % ProClin 300 20 C or at 2-8 C at 20 C: 1 year, repeated freeze / thaw cycles are possible at 2-8 C: 6 months
A7252
A7252,0125 125 ml A7252,0500 500 ml

Blocking Buffer III BSA saturates free binding capacities on surfaces of plastic consumables and other surfaces like ELISA plates and blotting membranes. Thus, a reduction of unspecific binding on surfaces can be achieved. Blocking Buffer III BSA is the standard surface blocker for many applications. If a blocker on basis of BSA (bovine serum albumin) is efficient enough for an assay, Blocking Buffer III BSA is the well-priced alternative to universally applicable and more complex blockers. Blocking Buffer III BSA can be used for ELISA, EIA, Western blotting, Immuno-PCR as well as protein arrays and immunohistochemistry. Immediately before use the buffer should be mixed thoroughly. Blocking Buffer III BSA is ready-to-use. Repeated freezing and thawing is possible. After immobilisation of capture antibodies or target proteins Blocking Buffer III BSA is applied without dilution in wells or on membranes. The incubation time has to be adopted depending on surface characteristics by the user. We recommend blocking over night at 4 C, but in many cases shorter incubation is also promising. After blocking, the surface has to be washed with Washing Buffer to prepare it for the next working steps. If you find just the same background or unspecific binding in spite of correctly used Blocking Buffer III BSA, we recommend the use of Blocking Buffer I (Prod.-No. A7099). Efficiency of blocking is significantly improved with Blocking Buffer I in comparison to standard blocking procedures by the special production method of this casein containing reagent, which leads to casein molecules varying in molecular sizes. This is achieved by chemical modification from highly purified casein. Therefore, you get a maximum of safety and reproducibility. Blocking Buffer I is suited for blocking of surfaces in all immunoassays, whereas Blocking Buffer III BSA leads to sufficient results in nonproblematic assays. Background and unspecific binding can not only occur at surfaces, but also between antibodies and components of complex specimen. In this cases only an assay buffer, which is used for the immunological detection reaction, can lead to satisfactory results. Therefore, we recommend to use CrossDown Buffer (Prod.-No. A6485) for optimal results in measurements of complex and important specimen. CrossDown Buffer has an effect like a filter for binding, which doesnt affect high affinity binding in any way, but depletes unspecific binding and interference like matrix effects and cross reactivities.
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Dream Teams
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products
Blocking Reagent CA
Blocking Reagent for Hybridization Assays and Western Blots
A3409,0010
Storage Room Temperature

The Blocking Reagent CA is used in hybridization and detection procedures using non-radioactive nucleic acid probes, and for Western blots. When immunoassays and hybridization assays, such as dot blots, Western blots, Southern blots, or Northern blots are performed, there is nonspecific binding resulting in high background. In order to reduce the nonspecific binding, Blocking Reagent CA is used to "block" unbound sites left after immobilization of the specific protein or after the hybridization with non-radioactive probe. The Blocking Reagent CA improves sensitivity and reduces background.
Note Nonfat dry milk inhibits the streptavidin-biotin interaction due to its content of biotin! Procedure
Proteins For blotting applications such as Western blots and dot blots, add 0.2 % (w/v) Blocking Reagent CA into TBST or PBST, heat to 75-80 C in a water bath or microwave oven, and stir well until dissolved. The Blocking Reagent CA dissolves to give a milky solution. Use for blocking and for dilutions of antibodies.
Note Do not use the Blocking Reagent CA in PBST for alkaline phosphatase conjugate dilutions
Nucleic Acids For hybridization applications add 0.2 % (w/v) Blocking Reagent CA to Tris-Saline buffer (100 mM Tris-Cl pH 7.5, 600 mM NaCl), heat to 60 -65C in a water bath or microwave oven, and stir well until dissolved. The Blocking Reagent CA dissolves to give a clear solution. Use for blocking after the wash steps, and before incu-bation in any enzyme-conjugate solution (e.g. Streptavidin-HRP, Streptavidin-AP).
Optimization of Time Required for Blocking with the Blocking Reagent CA

1. 2. 3. 4. 5. 6. 7. 8. 9. Cut 7 small squares of nitrocellulose or other suitable membrane. Label each square with a ball point pen in 10 minute increments (60, 50, 40, 30, 20, 10), and one without blocking. Place the first square (60) in a few ml of Blocking Reagent CA solution, and add successive squares at 10minute intervals. Wash all squares in TBST, PBST or Tris-Saline buffer (100 mM Tris-Cl pH 7.5, 600 mM NaCl). Dilute the secondary antibody or streptavidin (HRP-conjugated) in Blocking Reagent CA solution. Incubate on shaker for 1 hour. Rinse in TBST, PBST or Tris-Saline buffer three times, 10 minutes each time. Detect with the Chemiluminescent Detection Kit for horseradish peroxidase (Order-No. A3417,1200). Evaluate background intensity in each square. Select the incubation time that gives the lowest background.
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Coating Buffer C pH 9.6

Coating buffer for capture-antibodies and proteins on surfaces Composition pH value Preservative Storage Stability
A7150
A7150,0125 125 ml
Carbonate-based 10X stock solution pH 9.6 0.2 Buffer is delivered without any preservatives, because some preservatives can interfere with the process of coating. Thus coating buffer is safe and easy useable for many applications 20 C or at 2-8C Use working solution immediately! at 20C: min. 3 months at 2 -8C: 1 month

Coating Buffer C pH 9.6 is made for adsorptive immobilisation of proteins and antibodies on plastics surfaces (for example microtiter plates) or other protein binding surfaces. Applications are for example ELISA, EIA, RIA and protein arrays as well as immuno-PCR. Crystals of salt can precipitate during storage at 2-8 C or after freezing. Therefore Coating Buffer C must be warmed up to room temperature and should be mixed thoroughly before preparing the working solution. This leads to dissolving of salt after shaking. The stock solution is diluted 1:10 with deionized water to get the working solution. Use the working solution the same day. The proteins or antibodies for immobilisation are diluted in this working solution and used after mixing. The typical concentration range for standard ELISA is between 0.5 g/ml and 2 g/ml for capture antibodies. Depending on the surface as well as on proteins or antibodies the useful incubation times can differ. Consequently any user should optimise its own incubation procedure. For some proteins or antibodies Coating Buffer PBS pH 7.4 is better, for others Coating Buffer C pH 9.6 is advantages for immobilisation. The pH-value can have an influence on the steric structure of proteins or antibodies, thus having an effect on immobilisation. For an optimised procedure for a newly developed immunoassay we strongly recommend testing of both Coating Buffers in comparison.
Coating Buffer PBS pH 7.4

Coating buffer for capture-antibodies and proteins on surfaces
A7136
A7136,0125 125 ml
PBS-based 10x stock solution pH 7.4 0.2 Buffer is delivered without any preservatives, because some preservatives can interfere with the process of coating. Thus coating buffer is safe and easy useable for many applications 20C or at 2-8C at 20C: 1 year at 2-8C: min. 3 months

Coating Buffer PBS pH 7.4 is made for adsorptive immobilisation of proteins and antibodies on plastics surfaces (for example microtiter plates) or other protein binding surfaces. Applications are for example ELISA, EIA, RIA and protein arrays as well as immuno-PCR. Crystals of salt can precipitate during storage at 2-8 C or after freezing. Therefore Coating Buffer PBS pH 7.4 must be warmed up to room temperature and should be mixed thoroughly before preparing the working solution. This leads to dissolving of salt after shaking. The stock solution is diluted 1:10 with deionized water to get the working solution. Use the working solution the same day. The proteins or antibodies for immobilisation are diluted in this working solution and used after mixing. The typical concentration range for standard ELISA is 0.5 g/ml to 2 g/ml for capture antibodies. Depending on the surface as well as on proteins or antibodies the useful incubation times can differ. Consequently any user should optimise its own incubation procedure. For some proteins or antibodies Coating Buffer PBS pH 7.4 is better, for others Coating Buffer C pH 9.6 may have advantages for immobilisation. The pH-value can have an influence on the steric structure of proteins or antibodies, thus having an effect on immobilisation. For an optimised procedure for a newly developed immunoassay we strongly recommend testing of both Coating Buffers in comparison. 2010 AppliChem Immunoassay Buffer
27
products
CrossDown Buffer
Immunoassay buffer for minimisation of unspecific binding, cross-reactivities and matrix effects pH-Value Stabilizer Storage Stability pH 7.2 0.2 Phosphat-free, ready-to-use contains 0.1 % ProClin300 (ProClin is a registered trademark of Rohm and Haas) 20C or 2-8C at 20C: 1 year, repeated freeze/thaw cycles possible at 2-8C: 6 months
A6485
A6485,0050 50 ml A6485,0125 125 ml A6485,0500 500 ml

The newly developed CrossDown Buffer lowers cross reactivities, unspecific binding and matrix effects in immunoassays like ELISA, EIA, Western blotting, immuno-PCR, protein arrays, multianalyte immunoassays and immunohistochemistry depending on the characteristics of the assay type and the used antibodies. Mix the buffer thoroughly immediately before use. CrossDown Buffer is used instead of a sample buffer or antibody dilution buffer for the immunological reaction. CrossDown Buffer is not suitable for blocking of surfaces. For blocking of surfaces we recommend Blocking Buffer I (Order No. A7099). CrossDown Buffer is not suited as a sample buffer for electrophoresis.
Examples of use
ELISA dilution buffer for specimen and for the detection antibodies Western blotting dilution buffer for primary and secondary antibodies Immunohistochemistry dilution buffer for primary and secondary antibodies Protein arrays dilution buffer for specimen and for the detection antibodies we recommend the use of Oyster*- (Denovo Biolabels), CyDye**(Amersham) or Alexa***- (Molecular Probes) fluorescence dyes. We strongly recommend to test the effectiveness of CrossDown Buffer for a certain application. CrossDown in FACS analysis CrossDown buffer can replace the normally used FACS analysis assay buffer and is applied like the original assay buffer. In case that CrossDown is to active (i.e. reduction of the specific signal too), the most convenient way is to dilute the buffer with the original assay buffer (dilution 1 : 2 to 1 : 10). Alternatively, physiological buffers like PBS or Hepes can be used for diluting CrossDown.
*Oyster is a registered trade mark of the company Denovo Biolabels. **CyDye is a registered trade mark of the company Amersham Biosciences. ***Alexa Fluor Dye is a registered trade mark of the company Molecular Probes.
Dilution of the specimen Standards and specimen for ELISA and protein arrays can be diluted with CrossDown Buffer at 1:2 or higher. Standards and specimen should be treated strictly the same way. Dilution of antibodies Antibodies can be diluted with CrossDown Buffer in a user-defined manner, depending on the recommendation of the data sheet of the antibodies. This is the same for primary and secondary antibodies. Appearance of signal reduction In some cases a smooth reduction of the wanted signal can be observed. CrossDown Buffer reduces low- and middle-affinity binding. That means that by the use of lowand middle-affinity antibodies or polyclonal antibodies a smooth reduction of signals can appear. Polyclonal antibodies normally contain low- and middle-affinity binding components. In the case of polyclonal antibodies a moderate increase of the concentration of the antibody can lead to the previously seen signals. Unwanted low- and middle-affinity binding will be still reduced by CrossDown Buffer. In the case of low- and middle affinity antibodies (also monoclonal antibodies) a pre-dilution of CrossDown Buffer with salt-free water can be useful to get the previously seen signal. But in this case also the unwanted bindings or cross-reactivities can partly occur again, depending on the chosen dilution with water. Although CrossDown Buffer is used as an assay buffer it is necessary to saturate surfaces like ELISA-wells or membranes with a blocking agent. We recommend the use of Blocking Buffer I (Order No. A7099). CrossDown Buffer can be used additionally as a washing buffer especially in delicate or interference-sensitive assays like immuno-PCR. Components of immunoassays as well as of CrossDown Buffer may quench the fluorescence of fluorescein dyes. Therefore
Literatur [1] Miller, J.J. (2004) Clinical Laboratory International 28, (2), 14-17 [2] Kusnezow, W., Hoheisel, J.D. (2003) J. Mol. Recognit. 16, 165-176 [3] Patton, W.F. (2000) Electrophoresis 21, 1123-1144 [4] MacBeath, G. (2002) Nat. Genet. 32, 526-532 [5] Miller, J.J., Valdes, R.Jr. (1992) J Clin Immunoassays 15, 97-107 [6] Wood, W.G. (1991) Scand. J. Clin. Lab. Invest. Suppl. 205, 105-112 [7] Kricka, L.J. (1999) Clinical Chemistry 45 (7), 942-956 [8] Span, P.N., Grebenchtchikov N., Geurts-Moespot, J., Sweep, C.G.J. (2003) Clinical Chemistry 49 (10), 1708-1709
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FAQ
What's so special about CrossDown?
an antibody in an assay has to be adjusted very much depends on the quality of the antibody The newly developed CrossDown Buffer lowers cross reactivities, unspecific binding and matrix effects in immunoassays like ELISA, EIA, Western blotting, immuno-PCR, protein arrays, multianalyte immunoassays and immunohistochemistry. The specific, high affinity binding of antibody to analyte stays, while unwanted binding of the antibody is prevented.
Why does CrossDown Buffer assist in reducing matrix effects?
How do I use CrossDown Buffer?
CrossDown Buffer is used instead of sample buffer or antibody dilution buffer for the immunological reaction. The sample (e.g. human serum or plasma) and the detection antibody are diluted with CrossDown Buffer, depending on the source of the interference. Assuming nonspecific binding or cross reactivity of the detection antibody, dilution with CrossDown Buffer is recommended.
The primary cause of the so-called matrix effect is based on unwanted, low-affinity binding of matrix components (e.g. serum proteins in samples from human serum) to analytes or antibodies. The analyte may be masked by proteins or other components of the sample matrix, preventing the binding of the antibody to its target. CrossDown Buffer prevents masking and support binding of antibody and analyte. Antibodies may be masked by matrix components as well. CrossDown prevents this masking and reduces masking already present.
May I freeze-thaw the buffer several times?
Does CrossDown Buffer replace a blocking buffer for surfaces (e.g. Western blot membranes or ELISA plates)?
The buffer can be frozen and thawed several times without a problem. After thawing, the buffer has to be mixed thoroughly before application to guarantee uniform distribution of the components.
No! CrossDown Buffer is not suitable for blocking of surfaces, but used for dilution of the sample and/or assay antibodies instead. For blocking of surfaces we recommend Blocking Buffer I (Order No. A7099).
Is the buffer used diluted or undiluted?

CrossDown Buffer is a ready-to-use solution. The sample or the detection antibody can be diluted directly in CrossDown Buffer. In competitive assays and some applications in immunohistochemistry, it may make sense to dilute CrossDown Buffer with water or physiological buffer. The optimal conditions and ratios should be determined on a case by case basis.
Do I have to consider whether polyclonal or monoclonal antibodies are diluted with CrossDown?
No! CrossDown can be used in combination with both types of antibody preparations. In case of polyclonal antibodies, you have to keep in mind that CrossDown only supports high affinity binding. Low affinity binding is suppressed. Polyclonal antibodies are a mixture of antibodies with different affinities. Therefore it may be necessary to increase the concentration of a poylclonal detection antibody when used with CrossDown Buffer. Increasing the antibody concentration increases the concentration of high affinity antibodies as well, while the effect of low affinity antibodies will be minimized. Whether the concentration of
Do buffer components influence color reactions?

The enzymatic activity of alkaline phosphatase or peroxidase are not negatively influenced by CrossDown Buffer. In fact, the opposite is the case. Users report an increased enzyme activity, when labeled detection antibodies were incubated with CrossDown Buffer.
Can CrossDown be applied together with fluorescent dyes, e.g. in protein chip applications?
Yes. There are positive results in protein chip applications. In some applications using a few cyan-dyes (e.g. Cy5, Cy3, oyster dyes), an increased signal was observed.
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Why
CrossDown Buffer Interference effects Minimisation of interference regardless whether cross-reactivities, matrix effects or unspecific binding of assay components Minimisation of background Increase in reliability guarantees better results by avoiding interference For use in all immunoassays Antibody Diluent Background Quality of results No effects No positive effects on reliability Usability Usability with different detection methods Usable with all common detection methods, very good results with peroxidases, phosphatases and fluorescent labels Ease of application Ready-to-use Stabilisation of antibodies Assay antibodies are stabilised in CrossDown Buffer, even storage of antibodies in CrossDown is possible Positive effects, variations decrease, false results are avoided, validations can be passed successful and easy Cooling and freezing for long-time storage, repeated freezing and thawing possible, but no cooled transportation needed No effects on stability of assay antibodies Effects on validation (e.g. for new FDA-guidance for industry) Storage and transportation
using CrossDown Buffer??

HAMA-Blocker Minimisation only of interference derived from HAMAs (Human Anti Mouse Antibodies) All other interference effects lead to wrong results! Effects only if background comes from HAMAs Increase in reliability only when specimen / samples include HAMAs For use only for human specimen/ samples Usability depends on product, some only for use with peroxidases others only for use with phosphatases, negative quenching with fluorescent dyes has to be checked with some products Some products recommend pre-dilutions with other buffers No effects on stability of assay antibodies No minimisation of interference
Some products only for use in ELISA or Western blotting, many different specialised products Usability depends on product, some only for use with peroxidases others only for use with phosphatases, negative quenching with fluorescent dyes has to be checked with some products Some products recommend pre-dilutions with other buffers
No positive effects on validation, interference like matrix effects or cross-reactivities lead to high variations or false results Cooling or freezing for long-time storage, most products recommend cooled transportation
Positive effects only if HAMAs are inside the samples. Then false results can be avoided. No effect on matrix effects and other cross-reactivities Cooling or freezing for most products necessary, cooled transportation needed
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CrossDown Peroxidase-Stabilizer
Assay-diluent for one-step incubation assays peroxidase conjugate stabilizer l substitute for HAMA blocker
l l
products
A8625
A8625,0050 50 ml A8625,0125 125 ml A8625,0500 500 ml
pH 7.2 0.2 contains 0.1 % ProClin300 (ProClin is a registered trademark of Rohm and Haas) 2-8C. Do NOT freeze. May form a precipitate. 24 months
Description
In one-step incubation assays the specimen is incubated directly with the detector-conjugate saving additional washing steps. This is very convenient and enables for very fast assay protocols in comparison to sequential procedures. Unfortunately this significantly increases the risk of interference, matrix effects and problems due to HAMAs (human anti-mouse antibodies) and rheumatoid factors. For this reason reliability suffers in favour of user-friendliness. CrossDown Peroxidase-Stabilizer solves this problem. CrossDown Peroxidase-Stabilizer enables assays with the highest reliability and robustness by minimising interference. At the same time CrossDown Peroxidase-Stabilizer is a premium stabilizer for peroxidase conjugates. Therefore conjugates can be stored directly in very low ready-to-use concentrations. Thus convenience and reliability are no longer incompatible.

CrossDown Peroxidase-Stabilizer is a dilution buffer for long-term storage of peroxidase-conjugates. This can be antibody-peroxidase-conjugates as well as Neutravidin- or Streptavidin-peroxidase-conjugates. Conjugates can be diluted directly in CrossDown Peroxidase-Stabilizer to the final concentrations. Typical concentrations of conjugates are ranging from 40 to 500 ng/ml. Immediately before use the buffer should be mixed thoroughly. CrossDown Peroxidase-Stabilizer is a combination of CrossDown Buffer and a new stabilizer for stabilizing antibodies and peroxidase. Therefore you get long-term stabilization as well as the CrossDown Buffer effect in one solution. The CrossDown Buffer effect: CrossDown Buffer was specially developed to minimise cross reactivities, nonspecific binding and matrix effects in immunoassays. Interference derived from HAMA (human anti-mouse antibodies) rheumatoid factors or other endogenous interfering factors can be reduced significantly. Observation of signal reduction: In some cases a slight reduction of the wanted signal can be observed. CrossDown Buffer reduces low and medium-affinity binding. That means that by using low- and medium-affinity detector-antibodies or polyclonal detector-antibodies a smooth reduction of signals can appear. Polyclonal antibodies normally contain low- and medium-affinity binding components. In the case of polyclonal antibodies a moderate increase of the concentration of the antibody can lead to the previously seen signals. HRP long-term stabilization of peroxidase: CrossDown Peroxidase-Stabilizer contains stabilizing components for antibodies and for peroxidase. This makes CrossDown Peroxidase-Stabilizer the ideal solution, if you want to store peroxidase-conjugates for a long period in a ready-to-use solution. Dilute the conjugates directly in CrossDown Peroxidase-Stabilizer. Stabilization of a HRP-antibody conjugate by a competitor's enzyme stabilizer product was compared to stabilization by CrossDown Peroxidase-Stabilizer (see fig. 1). CrossDown Peroxidase-Stabilizer provides the best long-term stabilization of the HRP-antibody conjugate. Testing CrossDown Peroxidase-Stabilizer with any new assay is recommended. Stability data of one peroxidase-conjugate cannot be transferred to other conjugates. Therefore every conjugate has to be tested for its shelf life in CrossDown Peroxidase-Stabilizer. If CrossDown Peroxidase-Stabilizer is used for immunodiagnostic kits, the shelf life has to be tested according to the current European directive for invitro-diagnostics or according to corresponding rules, which apply.
Figure 1: Shelf life of HRP-antibody conjugate bound to the analyte is measured as peroxidase signal in an ELISA and given as % value of freshly diluted HRP-antibody conjugate. Detector-conjugate concentration was 40 ng/ml.
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products
Peroxidase-Stabilizer
pH value Preservative Storage Stability pH 7.2 0.2 contains 0.1 % ProClin300 2-8C. Do NOT freeze. May form a precipitate. 24 months
A8647
dilution buffer and storage solution for peroxidase-conjugates for an outstanding shelf-life of the conjugates A8647,0050 50 ml l protection and stability of your HRP-conjugate for years A8647,0125 125 ml l Peroxidase-Stabilizer maintains enzyme activity A8647,0500 500 ml l Peroxidase-Stabilizer maintains functionality of HRP-conjugate l conjugate may be stored at very low final concentration no pre dilution steps
Description
Stabilization of all active components is very critical to get reliable results with immunoassays. When assays are stored for long-term before use, it is especially important to ensure adequate stability. Peroxidase-Stabilizer works very well as a stabilizer. Additionally it is a very good assay buffer. This enables any user to store HRP conjugates directly in ready-to-use concentrations, even when these concentrations are very low. This saves pre-dilution steps before doing an assay. Peroxidase-Stabilizer maintains the activity of conjugates of antibodies and of Neutravidin conjugated to HRP. Therefore it can be used in any assay, in which HRP is the detector enzyme. Peroxidase-Stabilizer decreases the variability often caused by changing shipping and storage conditions, thereby producing more consistent results with your diagnostic test kit. Peroxidase-Stabilizer can be easily incorporated into most assay protocols by substituting it for your conjugate diluent.

Conjugates can be diluted directly in Peroxidase-Stabilizer in its final concentration. Typical concentrations of conjugates are ranging from 40 to 500 ng/ml. Peroxidase-Stabilizer can be used directly as assay buffer in immunoassays. Peroxidase-Stabilizer is designed for troublefree assays without interference. If you have background issues or observe false-positives for example derived from cross reactivities, matrix effects or HAMAs we recommend using CrossDown-Buffer or CrossDown Peroxidase-Stabilizer rather than Peroxidase-Stabilizer. Suitability of Peroxidase-Stabilizer for a specific assay has to be tested by the user. Immediately before use the buffer should be mixed thoroughly. Peroxidase-Stabilizer long-term stabilization of peroxidase: Peroxidase-Stabilizer contains stabilizing components for antibodies and for peroxidase. This makes Peroxidase-Stabilizer the ideal solution, if you want to store peroxidase-conjugates for a long period in a ready-to-use solution. Dilute the conjugates directly in PeroxidaseStabilizer.
Stability test
Stability stress tests were performed with Peroxidase-Stabilizer (PS) using a monoclonal antibody conjugate (see fig. 1). HRPconjugate of the antibody was stabilized either by a so far leading enzyme stabilizer product or by Peroxidase-Stabilizer. Stress tests were performed at 45C over a period of up to 143 days. Percent retained activity was determined by comparing the activity of the HRP-antibody conjugate bound to the analyte. After 143 days incubation at 45C with Peroxidase-Stabilizer the monoclonal antibody conjugate retained around 80 % enzymatic HRP activity. This shows a shelf life at 4C with approximately 80% enzymatic activity of more than 6 years by conservative correlation according to Arrhenius. This is a very good stability and therefore PeroxidaseStabilizer gives an optimum product safety for HRP containing immunoassays! Testing Peroxidase-Stabilizer with any new assay is recommended. Stability data of a peroxidase-conjugate cannot be transferred to another conjugate. Therefore every conjugate has to be tested for its shelf life in Peroxidase-Stabilizer. If Peroxidase-Stabilizer is used for immunodiagnostic kits, the shelf life has to be tested according to the current European directive for invitro-diagnostics or according to corresponding rules, which apply.
Figure 1: Shelf life of HRP-antibody conjugate bound to the analyte is measured as peroxidase signal in an ELISA and given as % value of freshly diluted HRP-antibody conjugate. Detector-conjugate concentration was 40 ng/ml.
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Sample Buffer TSample buffer and dilution buffer for antibodies for use in immunoassays and immunohistochemistry
A7101
A7101,0050 50 ml A7101,0125 125 ml A7101,0500 500 ml
detergent-free, phosphate-free pH 7.2 0.2 contains 0.1 % ProClin300 20 C or at 2-8 C; repeated freezing and thawing cycles possible at 20 C: 1 year ; at 2-8 C: 6 months

Sample Buffer T- is a dilution buffer for specimen and antibodies fr direct use in immunoassays. Antibodies, coupled to alkaline phosphatase or peroxidase, can be diluted and used in Sample Buffer T- without problems, because Sample Buffer T- is free of phosphate. Sample Buffer T- without detergents contains neither Tween nor other detergents. Thus it is for use especially in immunohistochemistry, where detergents sometimes can make problems in detection. Immediately before use the buffer should be mixed thoroughly. Sample Buffer T- is ready-to-use. Repeated freezing and thawing is possible. Primary and secondary antibodies can be diluted directly in Sample Buffer T-. In case you observe increased background or unwanted matrix effects in the presence of Sample Buffer T-, we recommend the use of CrossDown Buffer (Artikel-Nr. A6485) instead of Sample Buffer T-.
Sample Buffer T+
Sample buffer and dilution buffer for antibodies for use in immunoassays
A7134
A7134,0050 50 ml A7134,0125 125 ml A7134,0500 500 ml
contains Tween pH 7.2 0.2 contains 0.1 % ProClin300 20 C or at 2-8 C; repeated freezing and thawing cycles possible at 20 C: 1 year ; at 2-8 C: 6 months

Sample Buffer T+ is a dilution buffer for specimen and antibodies fr direct use in immunoassays. Antibodies, coupled to alkaline phosphatase or peroxidase, can be diluted and used in Sample Buffer T+ without problems, because Sample Buffer T+ is free of phosphate. Sample Buffer T+ can be used for ELISA, EIA or Western blotting as well as for immuno-PCR or protein arrays. Sample Buffer T+ is not for use as an electrophoresis buffer. Immediately before use the buffer should be mixed thoroughly. Sample Buffer T+ is ready-to-use. Repeated freezing and thawing is possible. Specimen with the analyte as well as the detecttion antibody is diluted in Sample Buffer T+ and then used in the assay. It is necessary to treat standards and specimen in the same way! In case you observe increased background or unwanted matrix effects in the presence of Sample Buffer T+, we recommend the use of CrossDown Buffer (Artikel-Nr. A6485) instead of Sample Buffer T+. 2010 AppliChem Immunoassay Buffer
33
products
Stripping Buffer I
Stripping buffer for Western blots for multiple reprobing Composition pH value Preservative Storage Stability doesn't contain -mercaptoethanol and DTT pH 2.8 0.2 contains 0.1 % ProClin300 20 C or at 2-8 C at 20 C: 1 year at 2-8 C: 6 months
A7140
A7140,0050 50 ml A7140,0125 125 ml

Ready-to-use Stripping Buffer I removes reaction solution, primary and secondary antibodies from Western blotting membranes. After stripping the membrane can be used for a second detection (reprobing) with the same or other antibodies. The membrane is incubated in a vessel with Stripping Buffer I for removing the antibodies. For this purpose gently shake the membrane in Stripping Buffer I for 30-60 minutes at room temperature. After stripping, the membrane has to be washed with Washing Buffer and can be used for a second detection. If you detect with alkaline phosphatase, the wash buffer should not contain phosphates. Important: The designated incubation conditions are standard values, which have to be adopted by the user. Incubation times are depending on characteristics of the used antibodies.
Washing Buffer TrisT- (10X)

Washing buffer for use in ELISA, EIA, Western blotting and immunohistochemistry
A7137
A7137,0500 500 ml
Tris-based 10X buffer with 350 mM NaCl; contains no detergents pH 7.2 0.2 contains 0.1 % ProClin300 20 C oder bei 2-8C Use working solution immediately! at 20 C: 1 year at 2-8 C: 6 months

This Washing Buffer TrisT- is made especially for immunohistochemistry. It should be used, whenever detergents can make problems in detection. Crystals of salt can precipitate during storage at 2-8 C or after freezing. Therefore Washing Buffer TrisT- must be warmed up to room temperature and should be mixed thoroughly before preparing the working solution. This leads to dissolving of salt after shaking. The stock solution is diluted 1:10 with deionized water to get the working solution. Use the working solution the same day.
34
Washing Buffer TrisT+ (10X)

Washing buffer for use in ELISA, EIA and Western blotting
A7158
A7158,0500 500 ml
Tris-based 10X buffer; contains Tween and 350 mM NaCl pH 7.2 0.2 contains 0.1 % ProClin300 bei 20 C or at 2-8C Use working solution immediately! at 20 C: 1 year at 2-8 C: 6 months

Washing Buffer TrisT+ is used for many immunoassays. Washing steps are needed to remove unbound and excessive components, which are able to interfere with the assay. Application fields are ELISA, EIA, RIA, Western blotting as well as immuno-PCR, protein arrays and multianalyte immunoassays. It is applicable in automatic plate washers depending on salt tolerance of the washer.
Caution Make sure that you have checked the specifications and instructions of your washer!
Crystals of salt can precipitate during storage at 2-8 C or after freezing. Therefore Washing Buffer TrisT+ must be warmed up to room temperature and should be mixed thoroughly before preparing the working solution. This leads to dissolving of salt after shaking. The stock solution is diluted 1:10 with deionized water to get the working solution. Use the working solution the same day. Especially for use in immunohistochemistry we offer Washing Buffer TrisT- without Tween or other detergents (Product No. A7137).
35
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Blocking Reagents Albumin acetylated Albumin Fraction V (pH 7.0) Blotting grade Denhardt's - Solution (50X) BioChemica Denhardt's powder mixture (for 50X stock solution) Dextran sulfate 500 sodium salt BioChemica Dextran sulfate 500 sodium salt Molecular biology grade Gelatin powdered pure Ph. Eur., NF Heparin sodium salt Nonfat dried milk powder Polyvinylpyrrolidone (K90) Molecular biology grade Salmon sperm DNA sodium salt Salmon sperm DNA sodium salt (sonified) Transfer membranes Reprobe Nitrocellulose supported 0.22 m Transfer membrane Reprobe Nitrocellulose supported 0.45 m Transfer membrane Pure Nitrocellulose unsupported 0.22 m Transfer membrane Pure Nitrocellulose unsupported 0.45 m Transfer membrane Pure Nylon Neutral Transfer membrane 0.22 m (30 cm x 3 m) Pure Nylon Neutral Transfer membrane 0.45 m Reprobe Nylon Positively charged Transfer membrane 0.45 m (30 cm x 3 m) PVDF-Star Transfer membrane 0.45 m Brochure Transfer Membranes Other Related Products Chemiluminescence Detection Kit for Horseradish Peroxidase Peroxidase fom horseradish EIA and Immunology Grade I Peroxidase fom horseradish EIA and Immunology Grade II Sodium azide pure Streptavidin ultrapure Thimerosal BioChemica Prod. No. A3417 A3615 A3771 A1430 A1495 A1278 Prod. No. A0845 A6588 A2248 A3792 A2250 A4970 A1693 A3004 A0830 A2260 A2160 A2159 Prod. No. A5237 A5242 A5250 A5239 A4399 A5248 A5255 A5243
Further reading
Arnold M. Raem & Peter Rauch (Hrsg.) Immunoassays 2007 Elsevier Spektrum Akademischer Verlag (ISBN 3-8274-1636-1) german language David Wild (Ed.): The Immunoassay Handbook. 3rd Edition. Elsevier Science Publishing Company, Amsterdam, Boston, Oxford 2005, ISBN 0-08-044526-8 Werner Luttmann, Kai Bratke, Michael Kpper, Daniel Myrtek: Der Experimentator: Immunologie. 2. Auflage. Spektrum Akademischer Verlag, Heidelberg 2006, (ISBN 3-8274-1730-9) german language
36
infotaining
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Gel Electrophoresis Size Marker
Ready-to-use DNA size marker and protein size marker for gel electrophoresis are available from many sources. Besides these standard products, AppliChem offers the full range of lyophilized DNA markers. The advantage of lyophilized marker is their outstanding stability. The long shelf life of more than 5 years is achieved by deproteinization and subsequent lyophilization. Read more about the usage of our markers in our new brochure.
Biological Buffers
Many biochemical processes are markedly impaired by even small changes in the concentration of free H+ ions. It is therefore usually necessary to stabilise the H+ concentration in vitro by adding a suitable buffer to the medium, without, however, affecting the functioning of the system under investigation. Biological buffers are an essential part of each experiment. Several aspects have to be taken into account, when planning an experiment. AppliChems brochure Biological Buffers imparts basic knowledge on the criteria for selecting the right buffer and requirements of buffers.
Detergents
Detergents are more than just air bubbles. As diverse as the research objects and techniques are as diverse are the detergents available. The properties of e.g. SDS and octylglucoside are so different that you hardly may exchange them for the identical experiment. Why do you apply SDS in gel electrophoresis but not dodecylmaltoside? Because your colleagues did it all the time before? AppliChems brochure Detergents will help you selecting the best detergent for your assay.
Transfer Membranes
AppliChem supplies a range of transfer membranes designed and tested specifically for RNA, DNA and protein analysis. AppliChem also provides our customers with tried and proven protocols developed to obtain consistent reproducible and dependable results when used with AppliChem membranes. 22 protocols and all types of membranes all from one source.
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Safety First: Banish Mycoplasma

The contamination of cells with mycoplasma is a very common problem, even though it often goes unnoticed since no cloudiness appears in the cell culture. Mycoplasma are small and may pass the filtration units applied for preparing cell culture media. They do influence the growth, morphology and survival of the host cells, resulting in a strong influence on the test system and the results obtained. AppliChem offers a PCR-based test kit to test for infection, antibiotics for the treatment of infected cells, and cleansing reagents for CO2-incubators and waterbaths to prevent infection.
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Applichem Immunoassay Buffer

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Immunoassay Buffer

Take the Pink Link!

Thinking without knowledge makes chance the ruler.

Take the Pink Link!

Werner Kollath, German bacteriologist

Probleme & praktische Lsungen

Ever since the foundation of the firm, AppliChem has

Gel Electrophoresis Size Marker

the market from the very beginning. Our growth confirms

Take the Pink Link!

the relevance of these measures.

Using ready-to-use ELISA

kits from manufactu

times however, home-ma

the right antibodies or the

days the plates d

Keywords NukleinsureDekontamination DNA-Degradationstest PCR-Test

A comprehensive catalogue of products, with detailed

AppliChem brings advantages through knowledge.

Size-Exclusion Chromato grap for purification of biomolec hy ules

DNA-freie Reagenzien und Mastermixe fr die PCR

It is indeed correct that smaller molecules

pass more slowly through t

2010 AppliChem Immunoassay Buffer

Limiting Cross Reactivity in Immunoassays

Immunoassay Buffer AppliChem 2010

2010 AppliChem Immunoassay Buffer

Immunoassay Buffer AppliChem 2010

Interference caused by endogenous components of the specimen

Immunoassay Buffer AppliChem 2010

2010 AppliChem Immunoassay Buffer

PBS/BSA-Standard buffer CrossDown Buffer

Immunoassay Buffer AppliChem 2010

2010 AppliChem Immunoassay Buffer

Without Blocking ... No Result!

Blocking Buffer I - ready-to-use

Immunoassay Buffer AppliChem 2010

Just theory? This is the practice!

with Standard Assay Buffer

2010 AppliChem Immunoassay Buffer

tips & tricks

Is it possible to store antibodies in 50 % glycerol?

Does it make sense to add albumin?

ELISA, EIA, RIA

Immunoassay Buffer AppliChem 2008

Washing Buffer TrisT+ Washing Buffer TrisTCoating Buffer Stripping Buffer I

Immunoassay Buffer AppliChem 2010

Nucleic Acid Decontamination with The ExitusPlus Technolo

Improving quality of ELISA

Size-Exclusion Chromatograph y for purification of biomole cules

times however, home-made the right antibodies or the

completely different story.

because after storage of some

Keywords chemical properties usefull pH range buffer preparation

Recommendations for the setting 1. Temperature

Practical Tips Preparing Buffer

of the pH value of a buffer and

Interfering effects storage conditions

Keywords improved resolution Polyacrylamide Gel Electrophoresis SeparateIT Polymer Solution

fluorescent labels available

top-notch brightness perfor-

cells that contain non-recombinant

blue/white screening is not

required for this system.