Introductory Laboratory

Experiments

Gunnel Dalhammar 1992,

Revised by Sissela Liljeqvist 1996, Malin Eklund 2000,
Marianne Larsen 2003, Marianne Larsen/Alex S Rajangam 2004 and
Alex S Rajangam 2005

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TABLE OF CONTENTS

Introduction to analytical biochemistry 3

Proteins 4

Protein analysis methods

1. Salt precipitation of Globulin and
Albumin from bovine serum 7

2. Protein content determination: 9

The Bradford method 9

3. Electrophoretic techniques:
Electrophoresis 11
SDS-PAGE 12
Western Blot 13

4. ELISA 19

Appendices:
1. Ammonium sulfate saturation table 25
2. Composition of human plasma 26
Protein molecular weight marker 26
3. Guidelines for writing the laboratory report 27

2
Introduction to Analytical Biochemistry

The objective of this laboratory exercise is to familiarize protein and few

methods for protein analysis. Using some protein chemistry methods, we
will separate and identify two of the main protein fractions in the whole
bovine serum from calf.

The laboratory exercise will include:

1. Salt precipitation enabled fractionation of globulin and albumin protein

sample from the whole calf-serum.

2. Protein content determination of the two protein fractions and of

whole serum. Bradford method will be used to determine the
concentration of the protein samples.

3. Electrophoretic separation of the protein sample with SDS-PAGE and

Coomasie staining of the gel to check the purity of the protein fraction
and a western blotting done with gene specific antibodies for albumin
and globulin protein fractions.

4. Determination of the specific concentration of albumin content in

whole serum and also in the albumin fraction using an immunochemical
method with gene specific antibody called as ELISA.

3
Proteins

Proteins are macromolecules essential for life, taking part in almost all
biological processes. The building blocks of proteins are amino acids. The
amino acids are linked by peptide bonds, to form a polypeptide chain that folds
into a protein. The molecular weight of a protein can vary from around 6 000
Dalton (Dalton = g/mole) to above 1 000000 Dalton.
The genetic code (DNA) codes for 20 different amino acids, that differs in size,
polarity, hydrophobicity, flexibility and charge. The order in which these amino
acids are linked together and how this string of amino acids folds into a three
dimensional structure give each protein its unique properties. The possible
combination of varied sequence order of these amino acids provides the nature
with a wide choice of proteins for a definite function.

The biological properties of proteins can be understood in terms of protein

structure, that is, the three-dimensional relationships between a protein's
component atoms. Four levels of structure determine the shape of proteins. The
primary structure of a protein is the amino acid sequence of the polypeptide
chain. The secondary structure of a protein refers to the local organization of
parts of the polypeptide chain. Amino acids which lie close to each other are
stabilized by hydrogen bonds, resulting in folding of the polypeptide backbone
into mainly two geometric arrangements: an -helix or a -strand, Fig.1.
The tertiary structure describes how amino acids, at a greater distance from each
other, combine to give a protein its three-dimensional appearance, Fig.2. A
protein containing two or more polypeptide chains has a quaternary structure,
which describes the arrangement of the individual amino acid chains. Every
such chain is called a subunit. Within a protein it is possible to identify different
domains. This term is used to describe structural units within the protein.

Fig.1 Secondary protein structure: -helix and three -strands

4
Fig 2: Tertiary structure of the protein phosphotransferase. Schematically
showing -helices in black and -strands forming a Sheet in grey.

Fig 3: From Amino acid to Protein.

Examples of protein function

Enzymes are proteins, which serve as biological catalysts, increasing the
reaction rates, sometimes by a factor of 1012. There are complex sets of
reactions in all life processes. For example Carbonic anhydrase is an enzyme

5
necessary for our survival. This enzyme converts CO2 into HCO3. CO2 is not
very soluble in blood making it impossible for the blood to transport CO2 from
the blood to the lungs. HCO3 on the other hand is very soluble in blood allowing
the body to efficiently transport HCO3 to the lungs where it is converted back to
CO2 allowing us to breath out. The same enzyme performs the conversion back
to CO2. The direction of the reaction depends on the substrate abundance. Other
proteins also serve as regulators of these reactions, both directly as enzymes and
indirectly in the form of chemical messengers known as hormones as well as
receptors for these hormones.

Proteins are also involved in transportation and storage of biologically important

molecules such as oxygen, metal ions and glucose. Oxygen is transported in the
blood with the help of a protein called hemoglobin. Iron is transported by
transferrin and stored in the liver as ferritin.

Certain specialized proteins build up our immune system. Immunoglobulins or

antibodies are proteins, which form an essential biological defense system in
higher animals. They recognize and bind specifically to foreign substances that
enter the body and thereby triggering the bodies defense system.

Receptor proteins are very specific in their function, for example nerve impulses
where specialized protein acts as the receptor molecule for acetylcholine, which
enables the transmission of nerve impulses at synapses.

Repressor proteins control the expression of genes in the cell and helps in the
regulation of protein production. They can inactivate specific segments of DNA
in the cell. If repressor proteins were absent, a cell would continuously produce
all of the proteins, which are coded for by the cell's DNA.

Proteins also have important passive roles. Ex. Collagen, found in human skin
and connective tissues. Collagen is a structural protein, which provides bones
and ligaments with their characteristic strength.

Any variation of a native protein's structure may cause the protein to denature. If
this happens, the chemical, physical and biological properties of the protein will
change. Organic solvents, strong acids or bases, heating, vigorous shaking or
high pressure can cause denaturation of protein. A reversibly denatured protein
can resume its original structure and function, whereas an irreversibly denatured
protein cannot.

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1. Salt precipitation of globulin and albumin in bovine serum

Certain salts precipitate proteins in aqueous solution. This "salting out" is

usually reversible. The properties of the protein such as ionic strength,
hydrophobicity, hydrophilicity etc., determine their solubility. At a certain salt
concentration, water molecules surrounding hydrophobic patches on the surface
of a protein are removed by the added salt ions. Exposed hydrophobic areas in a
polar solvent are not energetically favorable so these patches will interact with
each other, resulting in aggregation and precipitation.

The two main fractions of proteins in serum are globulin (antibodies) and
albumin. Globulin is insoluble in distilled water and slightly soluble in dilute
salt solutions and can be precipitated using 50%-saturated ammonium sulfate.
Albumin is readily soluble in water and in dilute salt solutions. Albumin
precipitates first when using saturated ammonium sulfate concentrations. The
difference in solubility of proteins is in this lab utilized to fractionate serum into
its two main components.

Procedure:

1. Dilute 1 ml bovine serum (BS = calf serum) with 4 ml water.

2. Add 5 ml saturated ammonium sulfate (this dilution enables 50 %

saturated ammonium sulfate) and mix gently by inverting the tube.
Vigorous mixing can result in foaming of the protein mixture. Allow this
to stand for 15 minutes.

3. Spin in the tabletop centrifuge at maximum speed for 15 minutes.

Carefully decant the supernatant with a Pasteur pipette and transfer into a
new tube and save the precipitate (globulin fraction).

4. Add solid ammonium sulfate to the supernatant (albumin) slowly with

much care until 100 % saturation is achieved. Use appendix 1 to
calculate the approximate amount of solid ammonium sulfate needed to
reach 100% saturation.

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 5. Mix by inverting the tube. Allow the solution to stand for 15 minutes.
Spin in the tabletop centrifuge at maximum speed for 15 minutes. Decant
the supernatant. Dissolve the precipitate, which is the albumin fraction,
in 1 ml of distilled water.

6. Wash the globulin precipitate by adding approximately 5 ml of half

(50%) saturated ammonium sulfate solution (dilute the pre-prepared
saturated ammonium sulfate for this step).

7. Resuspend the pellet gently and centrifuge at maximum speed for 15

min. Dissolve the globulin precipitate in 1 ml of distilled water.
(Globulin should not be soluble in distilled water, but there might be
some salt still remaining in the tube).

Fig 4: Flowchart of Protein precipitation

Bovine serum
+
H2O
+
Saturated Ammonium
sulfate

100% Saturation is achieved

by adding Ammonium sulfate
needed.
Supernatant
Albumin fraction Albumin protein precipitates
at 100% saturation.

Mixed and
Centrifuged

Pellet -Globulin fraction Centrifuged and resulting pellet

is Albumin protein fraction

Pellet resuspended in 50%

Saturated Ammonium sulfate
and centrifuged. Pellet
resuspended in water.

Globulin protein fraction

8
2. Protein content determination of whole serum, the
albumin and the globulin fraction

A number of different methods exist to estimate protein content. Measuring the

absorbency of aromatic amino acids is one method often used to estimate total
protein concentration. Aromatic residues absorb strongly at 280 nm and can
therefore be correlated to protein concentration. Every method for estimation of
protein concentration is subject to error because of the great diversity between
proteins. To minimize errors involved in protein estimation, it is important to
use a good reference standard.

Two different methods will be used for the determination of protein content in
Bovine whole serum (BS) and in the albumin and globulin fractions. The
Bradford method is described in the following section. Use the method for all
three samples (whole serum, the albumin and globulin fractions) and compare
the results. Get yourself familiarized with the protocol for this procedure before
starting the actual work.

The Bradford Protein Content Determination Method

This protein estimation method is based on binding of a dye, Coomasie Brilliant
Blue to proteins. The degree of binding to different proteins varies with the
number of basic amino acids in the protein. The cationic form of the dye, which
predominates in the acidic assay solution, has an absorption maximum of 465
nm. The dye binds to a protein as the anionic form, which has an absorption
maximum of 595 nm. The protein concentration of the sample is measured by
measuring the absorbance at 595 nm.

Materials:
Bradford Reagent (prepared by the assistants):
100 mg Coomasie Brilliant Blue G-250 dissolved in 50 ml 95% ethanol. The
solution is mixed with 100 ml of 85% phosphoric acid and made up to 1 l with
distilled water. The reagent should be filtered through Whatman No 1 filter
paper before storage at room temperature and will be stable for several weeks.
Slow precipitation occurs, and refiltration of the stored reagent is necessary
before use.

Protein standards: Bovine Serum Albumin at a concentration of 1 mg/ml in

distilled water is used as a reference standard. Store frozen.

9
Things to think about:
All dilution series should be made in duplicates, meaning you should
make two separate dilution series for each of your three samples (whole
serum, the albumin and globulin fraction)
To minimize pippeting error, do no pipette less than 5 l.
One could consider having at least two dilutions within the range of the
standard curve for each of the three samples. And could estimate the
mean protein concentration with all the dilutions within the range of the
standard curve.

Procedure:
Standard Reference:
Dilution series for BSA (from the stock provided by the lab assistant) is made
with distilled water, in duplicates. At least 5 different concentrations are made
between the range 0.1 to 0.5 mg/ml. The water is used in the place of protein
solution for to blank (ZERO) the spectrophotometer reading. Dilutions of the
proteins need to be carefully vortexed. The protein samples are also diluted
similar to dilution of the standard reference, just to make sure that at least two
points of the dilution series lies within the ranges of the slope of the standard
curve. The final volume of the dilutions can be made up to 0.2 ml even though
we would use only 0.1 ml for the assay. NOTE: Use Appendix 2 to theoretically
estimate the concentration of the protein in serum.

1. Add 0.1ml of the protein solution (from the dilutions) into a test tube and
add 5 ml of Bradford reagent. Mix thoroughly by vortexing.
2. Measure the absorbance at 595 nm after 5 min but before 1 hour.

Fig 5: Photo showing the color formation in Bradford

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Increasing concentration of Protein
i
3. Electrophoretic techniques: SDS-PAGE and western blot

Electrophoresis, the migration of ions in an electric field, is widely used for

analytical separation of Biomolecules. An inert substance such as paper or gel is used
as a solid phase and the liquid phase is a buffered salt solution. When an electric current
is applied to the solid phase, the buffer serves as a conductor. The migration of
Biomolecules as they relate to electrophoresis.

Basic principle of PAGE:

Application of an electric field, E, to a particle with a charge (q) will generate a force

After the particle accelerates it will reach a constant velocity (v). When the frictional
forces (vf, velocity times frictional coefficient-f) equal the electrostatic forces

The frictional coefficient, f, for a spherical particle of radius a is

The mass of a particle of radius a and density r is

The velocity of a charged particle is directly proportional to the strength of the applied
field (E), The velocity of a charged particle is proportional to the charge-to-mass ratio
(q/f).

What happens when an electric field is applied to a solution of charged proteins?

Negatively charged molecules (anions) migrate to the + terminal (anode)

"Anions migrate to the anode."
Positively charged molecules (cations) migrate to the -ve terminal (cathode)
Neutral molecules don't move.

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Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)

Polyacrylamide Gel Electrophoresis, PAGE is a biochemical method used to

separate and determine the size of proteins. The solid phase is a polyacrylamide
gel, which is cross-linked with N, N methylene-bis acrylamide. The pore size of
the gel will vary with the concentration of polyacrylamide and the degree of
cross-linking. One can cast gels either in tubes or between two glass plates (a
slab gel).

SDS-PAGE is a commonly used variant of PAGE. SDS (sodium dodecyl

sulphate) is a negatively charged detergent, which both opens up the structure of
the protein and provides it with a strong negative net charge, distributed evenly
on the polypeptide chain. The charge that SDS treatment gives to a protein is
directly proportional to the size of the protein, except for very hydrophobic
proteins. This means that the ratio: Q/r is constant. Therefore, if the pore size in
the gel were large all of the proteins would move through the gel at an equal
rate. However, increasing the concentration of polyacrylamide or otherwise
reducing the pore size by increased cross-linking of the gel, will cause the
molecules to be separated on the basis of their size.
Fig 6: Chemistry of SDS and illustration of disulphide bonds in protein.

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Fig7: Set up of a common PAGE- Electrophoresis apparatus.

Western blot

The procedure for transferring separated proteins on a PAGE, to a nitrocellulose

membrane is called a "Western blot". Western blotting utilizes enzyme labeled
antibodies to identify specific proteins; this variation of the method is called as
Immunoblotting. The proteins are first separated using SDS-PAGE and then
electrophoretically transferred to a nitrocellulose membrane. The bound proteins
are probed with specific antibodies, which in turn are detected with anti-
antibodies that are labeled with the enzyme alkaline phosphatase. The protein-
antibody complexes are visualized through addition of an enzyme substrate that
develops a visible color complex.

Originally, a researcher named Southern, described a method of separating DNA molecules

electrophoretically with subsequent transfer to a nitrocellulose membrane filter. This method,
the "Southern blot", was named after its originator. Shortly after, an analogous method for RNA
was developed, called the "Northern blot". The analogous protein blotting method is called the
"Western blot".

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Fig 8: Diagrammatic illustration of the western blotting.

Objective:

You will learn how to perform an SDS-PAGE electrophoresis, which is a very

common and powerful method to visualize the protein content of a sample.
The protein mixture is separated by size and you can estimate the size of your
sample proteins by using a molecular weight marker. The marker is a mixture of
proteins with known molecular weights, see appendix 2. You will also learn how
to perform an Immunoblotting for specific detection of a protein.

Procedure:

1. Prepare the samples by diluting bovine serum, the albumin and globulin
fractions 5 times in distilled water. Transfer 20 l of each dilution to an
eppendorf tube. Add 5 l of the 5x-sample reduction mixture to each tube.
Heat to 95C for 10 minutes in a heating block and then spin down the
samples in a centrifuge for a few seconds.

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2. Prepare the gel by removing the cover with a pair of scissors and rinse the
plastic gel cassette in distilled water. Remove the strip at the bottom. Use
gloves while working with the gel!!!

3. Attach the cassette to prepare the upper buffer chamber. (The buffer standing
inside the chamber is the upper buffer, through which the electricity flow to
the gel) Two gels can be attached per container.

4. Fill the upper buffer container with electrophoresis buffer. The level of the
buffer should be between the upper and the lower glass plates, covering the
wells. Make sure that the chamber is not leaking!

5. Add electrophoresis buffer to the lower buffer chamber (This is the buffer
standing outside the cassette chamber and to which electricity pass out from
the gel. The buffer should cover about 1 cm of the glass plates, covering the
lower opening of the gel

6. Load 5 l of each sample per well. Organize the gels according to the
instructions given by the laboratory assistants.

7. Connect the electrodes to the power supply. Run the system at 150 volts
until the blue stain has passed through the gel.

8. Turn the power supply off and disconnect the electrodes. Disassemble the
blot apparatus and take the gel cassette apart by cutting along the black lines.
Cut the gel in 3 parts according to instructions.

9. Fix and stain one part of the gel. Use the two other parts of the gel to make a
Western blot.

Fig 9: Example PAGE- SDS gel stained with coomasie blue.

1 2 3 4 5 6 Lane 1: Molecular weight

marker useful to calculate the
size of the protein band.

Lane 2- 4: Impure protein

fractions.

Lane 5-6: Relatively pure

protein.

15
Procedure, Western blot:

10. The gel should be packed with the Nitrocellulose membrane; the direction of
flow of the current is from the gel to the membrane (like a sandwich with the
filter paper on both sides). Label the nitrocellulose membrane by cutting the
corners of the membrane according to instructions. Use gloves!
11. Attach the sandwich to the container (two in each Fig).
12. Add ice block and transbloting buffer to the container. The buffer should
cover the sandwiches.
13. Run the Western blot at 100 V (250 mA) with cooling for 1 hour.
14. Disassemble the blot apparatus and remove the filter. Soak the membrane in
blocking buffer (1% milk powder in PBS) for 15 minutes.
15. Wash the filter once in PBST (PBS buffer containing 0.05% Tween 20).
(Tween is a detergent, which reduces the unspecific protein-protein
interactions, and washes away excess antigen).
16. Cut the membrane in two. Make sure that they are both labeled.
17. Incubate one membrane with rabbit anti-BSA (1:500) and the other
membrane with rabbit anti-BGG (1:500) respectively, diluted in PBS, for 30-
45 minutes. Wash 3 times in PBST.
18. Incubate with anti-rabbit IgG-ALP (1:10 000), an antibody conjugated to the
enzyme alkaline phosphatase, diluted in PBS for 30-45 minutes. Wash 4
times in TST.
19. Add substrate buffer (1 substrate tablet dissolved in 10 ml of distilled water,
5 ml for each membrane). Develop for about 5 to 20 minutes. Terminate the
development by washing the membrane in distilled water. Use gloves!

Fig 10: Example for a Western blotting where a protein band seen in a
membrane with increasing intensity of the protein band with the
increasing concentration of the protein.

Protein detected with

its specific antibody

16
 Fig 11: Illustration for making the Gel cassette for western blotting:

Required Materials:

Electrophoresis equipment: Bio-Rad Criterion

Transbloting equipment: Bio-Rad Criterion Trans-blot cell Power supply
Criterion precast 12.5% Tris-HCl gels
Criterion 0.45 m Nitrocellulose membrane
Criterion Filter paper

Reagents for SDS-PAGE slab gels, prepared for you by the assistants:
1. Polyacrylamide/bisacrylamide 37.5 : 1.
2. 1.5 M Tris-HCl, pH 8.8.
3. 10% SDS.
4. TEMED.
5. 10% ammonium persulfate.

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6. 5x sample reduction mixture: 4.0mL dist-water, 1.0mL 0.5M Tris pH
6.8,0.8mL glycerol, 1.6mL 10%(w/v) SDS, 0.4mL beta-mercapto
ethanol, 0.2mL 0.05%(w/v) bromophenol blue
7. 5x electrophoresis buffer, pH 8.3: 15 g/l Tris, 72 g/l Glycine, 5 g/l SDS
Combine 60 ml of this mixture with 240 ml of distilled water before
electrophoresis.
Transblot Buffer, pH 8.3 25 mM Tris, 192 mM glycine, 20% MeOH

Fixing solution 5% glycerol, 10% HAc, 85% H2O.

Stain Coomasie Brilliant Blue R-250, diluted in the

fixing solution.

Destaining Solution 40% EtOH, 10% HAc

PBS: Phosphate Buffered Saline

100 mM PO43-, 150mM NaCl,

TBS Tris Buffered Saline

Tris Buffer 50 mM, pH 8.6, NaCl 150 mM,

Blocking Buffer Powdered milk solution, 1 g/100 ml PBS

PBST PBS with 0.05% Tween 20

TST TBS with 0.05% Tween 20

Development Buffer Tablets, dissolved in 10 ml of water:

Tris buffer 100 mM, 5 mM Mg2+
BCIP (5-bromo-4-chloro-3-indolyl phosphate)
0.15 mg/ml
NBT (nitro blue tetrazolium) 0.30 mg/ml

Antibodies:
Rabbit anti-BS = anti-bovine whole serum antibody
Rabbit anti-BSA = anti-bovine serum albumin
Rabbit anti-BGG = anti-bovine gamma globulin
Goat anti-rabbit IgG-ALP = antibody conjugated to alkaline phosphatase

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4. ELISA (Enzyme Linked Immuno Sorbent Assay)

The enzyme linked ImmunoSorbent assay ELISA, is a very sensitive

immunological method. The method was originally developed in Sweden and is
now considered a standard immunological method. It makes use of specific
antibodies and can be used to quantify antibodies as well as antigens (the
proteins that are recognized by the antibodies). This method allows you to
determine the concentration of a specific protein, as compared to the other
methods used this far that determine total protein concentration.

To quantify antibodies:

The first step of this assay is the binding of the

antigen to a solid phase. The solid phase is
usually composed of polystyrene.

In the second step, antibodies are bound to the

antigen.
The amount of binding of the antibodies can
be quantified in several ways.

One-way is to introduce a third step by

binding an enzyme-labeled antibody. The
enzyme label is covalently bound to the
secondary antibody. Presence of the bound
conjugate is demonstrated by addition of an
enzyme substrate, which produces a colored
product. The amount of color produced is a
measure of the amount of enzyme present,
which in turn is a measure of the amount of
primary antibody bound to the antigen.

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To quantify antigens:

Antigens, proteins that are specifically

recognized by an antibody can be quantified in
a variety of different ways.

a) Antigen-specific antibodies can be bound to

the solid phase.
Followed by the addition of the antigen to be
quantified.
The third step is addition of enzyme labeled
anti-antigen antibodies. The concentration of
antigen in the original sample is then
determined calorimetrically.

b) A second strategy for antigen quantification

is to bind a fixed amount of antigen to the
solid phase. The sample is added to the wells,
together with a known amount of its
corresponding specific antibody.
"Competition" between the bound and the free
antigen occurs. If there is a lot of free antigen,
less amount of antibody will be bound to the
solid phase. As before, the bound antibody can
now be detected using an enzyme labeled
antibody, a conjugate.

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Objective:

The objective in this laboration is to demonstrate the ELISA method. A (variant)

of the ELISA method will be used for determination of the concentration of
albumin in the albumin fraction. The procedure will be done in a slightly
modified way as compared to the instruction given in the introduction of this
chapter.

General description of the laboration:

First, a standard curve is made with different, known concentrations of BSA

coated on the microtiter plate.

Dilutions of the albumin fraction are also transferred to the microtiter plate, and
the proteins are allowed to bind to the surface of the plate during an overnight
incubation. The plate will now be coated.
After the overnight incubation, excess of protein is washed off with washing
solution. The solution contains Tween 20, a detergent, which reduces non-
specific interactions to the plastic surface.
After incubation with a blocking solution, the next step will be to incubate with
an antibody directed against BSA (called as Primary antibody). The antibody
will be diluted and transferred to all wells that are coated with a sample. After
incubation for one hour and washing, the amount of antibody bound to the plate
will be determined.

This is done with the secondary antibody produced in goat, directed against
conserved domain of IgG antibodies and will bind to the primary antibody.
This goat-antibody is conjugated to an enzyme, alkaline phosphatase.
After incubation and additional washes, the enzyme substrate, p-nitro phenyl
phosphate is added. A yellow colored product will be produced. The intensity of
the color, as determined spectrophotometrically at 405 nm, reflects the amount
of rabbit anti-BSA antibody present and indirectly the amount of BSA.

Plotting the absorbance at 405 nm against the log of the concentration of BSA in
the standard will give a standard curve.
Plotting the absorbance at 405 nm against the log of the dilution of the standard
BSA and the albumin fraction gives a curve that shows the quality of the ELISA
assay. The curves will have the following approximate appearance:

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 Abs.

Log dilution

Abs.

Log conc. ng/ml

Materials, prepared by the assistants:

Antigen 1: BSA, 10 mg/ml (for the standard curve)

Antigen 2: the albumin fraction

Antibody: rabbit anti-BSA (will be provided by the assistant)

Conjugate: goat anti-rabbit IgG-ALP diluted 1/1000 (will be provided
by the assistant)
ELISA plate: plastic plates with high binding properties

Coating buffer: 50 mM sodium carbonate, pH9.6

Incubation buffer: PBS (phosphate buffered saline), pH7.2
with 0.05% Tween 20 (v/v) = PBST
Washing solution: PBST

Enzyme substrate: p-nitro phenyl phosphate 1.25mg/ml (dissolved in water)

22
 Substrate buffer (1M dietanolamine buffer, pH9.8
containing 0.5 mM MgCl2) will be made (1:1) by the
assistants immediately prior to use.
Multi-channel pipettes

Procedure:

NOTE: All dilutions are made in coating buffer!

It is important in steps 1,2 and 3 that you work with greatest possible accuracy.
Do not proceed to step 3 until all dilution series are prepared. The dilutions have to be
prepared separately before addition to the ELISA plate.
Make duplicate samples (two separate dilution series).

Prepare the samples included in the standard curve. Dilute BSA IN COATING
BUFFER to final concentrations of: 1mg/ml, 10 g/ml, 1 g/ml, 500 ng/ml, 100 ng/ml,
50 ng/ml, 10 ng/ml, 5 ng/ml, 1 ng/ml, 0 ng/ml

Prepare the dilutions of the albumin fraction. Dilute the albumin in COATING
BUFFER. The solution series should include dilutions: 1/10, 1/50, 1/100, 1/500, 1/103,
1/5x103, 1/104, 1/5x104, 1/105, 1/5x105, 1/106, 1/5x106, 1/107, 1/5x107, 1/108, 1/5x108.

Dilution 5 time 2 time 5 time 2 time 5 time 2 time 5 time 2 time 5 time 2 time 5 time 2 time 5 time 2 time 5 time
factor

Dilution 1/10 1/50 1/100 1/500 1/103 1/5x103 1/104 1/5x104 1/105 1/5x105 1/106 1/5x106 1/107 1/5x107 1/108 1/5x108
Series

1. Transfer 100 l of all samples to a microtiter plate. Do not use the wells closest to
the edge of the plate. Allow the plate to stand in room temperature over night.

2. Wash the coated ELISA plate 3 times in washing solution (PBST).

3. Add blocking solution (0,5% gelatin in PBS) to all coated wells.

23
4. Incubate in room temperature for 30 minutes.

5. Wash the coated ELISA plate 3 times in washing solution (PBST).

6. Add 100 l anti-BSA antibody diluted 1/2000 in incubation buffer (PBST) to all
treated wells. The assistant will provide the diluted antibody!

7. Incubate in room temperature for 30 minutes.

8. Wash the coated ELISA plate 3 times in washing solution

9. Add 100 l Alkaline-Phosphatase conjugated goat anti-rabbit antibody diluted

1/1000 times in incubation buffer (PBST) to all treated wells. The assistant will provide
the diluted antibody!

10. Incubate in room temperature for 30 minutes.

11. Wash the coated ELISA plate 3 times in washing solution

12. Add 100l enzyme substrate solution, p-nitro phenyl phosphate, to all treated wells.

13. Read the absorbance at 405 nm in the ELISA plate reader.

Calculations: (Use an MS Excel sheet to plot the graph)

1. Standard curve: Plot the Absorbance to log dilution of the BSA on a graph
sheet. The plot is made for all the time points, value from the Elisa result.
2. The best time point with smooth negative sigmoidal curve is chosen and the
graph is made for its respective the absorbance to log concentration values. This
graph ends up in a smooth positive sigmoidal curve
3. The slope is calculated for the resulting Absorbance Vs Log concentration and
this slope should be used to calculate the concentration.
4. The time point used to plot Abs. Vs Log conc. for standard curve is used for the
experiment sample also.
5. The mean concentration of all the dilution in the particular time point is
calculated.

24
Appendix 1: Ammonium sulfate saturation table

Final concentration of ammonium sulfate, % saturation

10 20 25 30 33 35 40 45 50 55 60 65 70 75 80 90 100
Grams solid ammonium sulfate to be added to 1 l of solution
0 56 114 144 176 196 209 243 277 313 351 390 430 472 516 561 662 767
10 57 86 118 137 150 183 216 251 288 326 365 406 449 494 592 694
20 29 59 78 91 123 155 189 225 262 300 340 382 424 520 619
25 30 49 61 93 125 158 193 230 267 307 348 390 485 583
30 19 30 62 94 127 162 198 235 273 314 356 449 546
33 12 43 74 107 142 177 214 252 292 333 426 522
35 31 63 94 129 164 200 238 278 319 411 506
40 31 63 97 132 168 205 245 285 375 469
45 32 65 99 134 171 210 250 339 431
50 33 66 101 137 176 214 302 392
55 33 67 103 141 179 264 353
60 34 69 105 143 227 314
65 34 70 107 190 275
70 35 72 153 237
75 36 115 198
80 77 157
90 79

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 Appendix 2: Composition of human plasma
Protein molecular weight marker

Physical Characteristics of Selected Blood Proteins

Protein Molecular Sedimentation Density-1 pI Concentration in
3
Weight (kDa) Constant (S)* (cm /g) Plasma (mg/ml)
Immunoglobulin M 1000 18-20 0.723 5.1-7.8 0.8-0.9

2-Macroglobulin 820 19.6 0.735 5.4 2.65 (men)

3.35 (women)
High-density lipoprotein 435 5.5 1.093 - 0.37-1.17
Factor I (fibrinogen) 341 7.63 0.723 5.8 2-6
Factor XIII (fibrinase) 320 9.9 0.730 - -
Immunoglobulin G 153 6.6-7.2 0.739 5.8-7.3 12-18
Ceruloplasmin 134 7.1 0.714 4.4 0.27-0.39
Plasminogen 81 4.2 0.715 5.6 0.48
Lactoferrin 77 4.93 0.716 8.2-9.2 0.004
Transferrin 76 4.9 0.725 5.2 2-4
Factor II (prothrombin) 70.2 5.11 0.719 3.8 0.11-0.23
Serum albumin 69 4.6 0.733 4.9 35-45
Transcortion 51.7 3.79 0.708 - 0.041
Orosomucoid 44.1 3.11 0.675 2.7 0.75-1.0
Retinol-binding protein 21 2.3 0.720 4.4-4.8 0.04-0.06
Lysozyme 15 2.19 0.721 10.5 0.005

2-Microglobulin 11.5 1.6 0.727 - 0.0013

-13
*S=svedberg unit=10 s.

Low Molecular Weight [LMW] Calibration Kit

The Protein Mixture in each vial consists of:

Subunit Mol. Wt. Ref. Source
Phosphorylase b 94,000 J Rabbit muscle
Albumin 67,000 I Bovine serum
Ovalbumin 43,000 I Egg white
Carbonic anhydrase 30,000 K Bovine erythrocyte
Trypsin Inhibitor 20,100 L Soybean
-Lactalbumin 14,400 M Bovine milk

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Appendix 3: Guidelines for writing the laboratory report

The report should be written in Swedish or in English.

The report should be written and organized in a way, which makes it possible for the
reader to understand the objectives and the results without having to consult the
laboratory manual.

Describe the basic principles of the methods used, present and interpret the results.

All primary data should be enclosed with the report, for example your calculations used
when preparing your dilution series (they do not need to be typed) and your ELISA
printouts used.

Compare the results from the different protein content determination methods and
explain similarities and differences between them.

Compare the Coomasie staining and the Immunoblotting of electrophoretically

separated proteins. Explain how the methods differ in specificity and sensitivity.
Remember that the antibodies might give rise to more than one band on the gels and
explain why that is so.

Analyze the results from the electrophoresis experiments. Identify the protein bands and
determine their molecular weights.

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