Proteomics terms

Bovine serum albumin

• also known as "Fraction V“ because The nickname
"Fraction V" refers to albumin being the
fifth fraction of the original Edwin Cohn purification
methodology that made use of differential solubility
characteristics of plasma proteins.
• is a serum albuminprotein derived from cows.
• is often used as a protein concentration standard in
lab experiments.
• mature BSA protein that contains 583 amino acids.
• Molecular weight: 66,463 Da (= 66.5 kDa)
 What is BSA standard curve?
• To know determine the actual concentration
of a protein a standard curve is required.
• A standard curve is a plot of absorbance vs. a
varying amount of some known concentration
of protein.
• Two common proteins used for standard
curvesare bovine serum albumin (BSA) and an
immunoglobin (IgG).
 Is BSA water soluble?
• Albumins are readily soluble in water and can
only be precipitated by high concentrations
of neutral salts such as ammonium sulfate.
Sigma tests the solubility of powdered BSA
in deionized water at 40 mg/mL and obtains
clear to very slightly hazy, faint yellow
solutions.
 Y used for standard?
BSA is used because of
its stability to increase signal in assays,
its lack of effect in many biochemical reactions,
its low cost, since large quantities of it can be
readily purified from bovine blood, a
byproduct of the cattle industry.
 How do u make 3% BSA?
• A simple way of making a 1% BSA solution is
to weigh out one gram of BSApowder, pour it
into a graduated cylinder that can hold more
than 100 milliliters (mL) of water, and then
add water until the liquid level reaches the
100 mL mark. Mathematically, one divided by
100 equals 1%
 What is stranderd curve for protein?
• Standard curves represent the relationship
between two quantities. They are used to
determine the value of an unknown quantity
(glucose concentration) from one that is more
easily measured (NADH level).
 How do u dissovle BSA?
• Bovine serum albumin (BSA) For a 10% (100
mg/mL) stock solution of BSA,dissolve 1 g
powdered Fraction V or molecular biology
grade BSA in 10 mL of distilled H2O; to avoid
clumping, dissolve by layering the powder on
the surface of the liquid. Gently rock the
capped tube until
the BSA has dissolved completely.
• BSA precursor protein is 607 aa.
• N-terminal 18-residue signal peptide is cut off
from the precursor protein upon secretion,
• hence the initial protein product contains 589
amino acid residues.
• An additional four amino acids are cleaved to
yield the mature BSA protein that contains
583 amino acids
• amphoteric compound is a molecule or ion that can react both as an acid and as
a base.
• Many metals (such as copper, zinc, tin, lead, aluminium, and beryllium) form
amphoteric oxides or hydroxides.
• Amphoterism depends on the oxidation statesof the oxide. Al2O3 is an example of
an amphoteric oxid.
• amphi-, which means both.
• amphoteric substance is a substance that has the ability to act either as an acid or
a base.
• Remember that acids donate protons (or accept electron pairs) and bases accept
protons.
• Amphoteric substances can do either. Just like a double agent can act as an ally or
an enemy, an amphoteric substance can act either as an acid or a base depending
on what substance it is reacting with.
• will exist mostly as zwitterions in a certain range of pH. The pH at which the
average charge is zero is known as the molecule's isoelectric point. Ampholytes are
used to establish a stable pH gradient for use in isoelectric focusing.
 isoelectric point (pI, pH(I), IEP),
• is the pH at which a particular molecule carries no net electrical
charge.
• is electrically neutral in the statistical mean.
• The standard nomenclature to represent the isoelectric point is
pH(I),
• although pI is also commonly seen.
• The net charge on the molecule is affected by pH of its surrounding
environment and can become more positively or negatively charged
due to the gain or loss, respectively, of protons (H+).
 What is IEP of aa?
• Amino acids can exist as zwitterions
containing a protonated amine group and
deprotonated carboxyl group.
• The pI, or isoelectric point, corresponding to
the zwitterion form lets you calculate the pH
at which an amino acid will have a net zero
charge
 How does ISP affect solubility?
• The surface of a protein has a net charge that
depends on the number and identities of the
charged amino acids, and on pH.
• This pH is called the isoelectric point, and for
most proteins it occurs in the pH range of 5.5
to 8. A protein has its lowestsolubility at
its isoelectric point.
• If the pH is less than the pI, the amino acid
will move toward the negative electrode. If
the pH is greater than the pI, the amino acid
will move toward the positive electrode. If
the pH is less than the pKa of a group, the
predominant form will be the conjugate acid.
 Why does protein precipitate in IEP?
• the isoelectric point (pI) is the pH of a solution
at which the net charge of a protein becomes
zero.
• At solution pH that is above the pI, the surface
of the protein is predominantly negatively
charged, and therefore like-charged molecules
will exhibit repulsive forces.
 Are pKa & ph same?
• The pKa is a measure of the strength of an acid. It depends on the identity
and chemical properties of the acid.
• Specifically, it's the negative log of the dissociation constant for an acid in
water.
• There is no way to convert pKa to pHbecause they're not equivalent
• The pH is a measure of acid in solution.
• pKa is an equilibrium constant.
• pH is an indication of hydrogen ion content in a solution.
Any changes to pH will therefore affect one of the factors in
the pKaequation.
• The amount and direction of change in the pKa value will depend on
whether the H+ ions are part of the reactant or product side of the
equation.
 What is pKa vakue of aa?
• pKa is the negative base-10 logarithm of the acid
dissociation constant (Ka) of a solution. The lower the
pKa value, the stronger the acid. For example, the pKa
of acetic acid is 4.8, while the pKa of lactic acid is 3.8.

Ka constant for acetic acid (CH3COOH) is 0.0000158 (= 10-4.8), but the pKa constant
is 4.8, which is a simpler expression. In addition, the smaller the pKa value, the
stronger the acid.

pKa is a property of a compound that tells us how acidic it is. The lower the pKa,
the stronger the acid. pH is a property of a particular solution that depends on the
concentrations and identities of the components. ... Acids are neutral when
protonated and negatively charged (ionized) when deprotonated.
 Sephadex
• is a trademark for cross-linked dextran gel
• used for gel filtration.
• was launched by Pharmacia in 1959, after development work by Jerker Porath and Per Flodin.
• The name is derived from separation Pharmacia dextran.
• It is normally manufactured in a bead form and most commonly used for gel filtration columns.
• By varying the degree of cross-linking, the fractionation properties of the gel can be altered.

is used to separate low and high molecular weight molecules.

is a faster alternative to dialysis (de-salting),
requiring a low dilution factor (as little as 1.4:1),
with high activity recoveries.
 is also used for buffer exchange and the removal of small molecules during the
preparation of large biomolecules, such as ampholytes, detergents, radioactive or
fluorescent labels, and phenol (during DNA purification).
Sephadex,
Sepharose,
Sephacryl (Molecular Biology)

are trade names for three types of gel-filtration matrices made

by the Pharmacia Fine Chemicals Company (1).

Sephadex is a bead-formed gel prepared by cross-linking dextran with

epichlorohydrin.
• These highly specialized gel filtration
and chromatographic media are composed of
macroscopic beads synthetically derived from
the polysaccharide dextran. The organic
chains are cross-linked to give a three-
dimensional network having functional ionic
groups attached by ether linkages to glucose
units of the polysaccharide chains.
 Dextran

is a complex branched glucan (polysaccharide derived from the condensation

of glucose.

 "Branched poly-α-d-glucosides of microbial origin having glycosidic bonds

predominantly C-1 → C-6".

Dextran chains are of varying lengths (from 3 to 2000 kilodaltons).

The polymer main chain consists of α-1,6 glycosidic linkages between glucose
monomers, with branches from α-1,3 linkages.

 This characteristic branching distinguishes a dextran from a dextrin, which is a

straight chain glucose polymer tethered by α-1,4 or α-1,6 linkages.
Epichlorohydrin

is an organochlorine compound and an epoxide.

Despite its name, it is not a halohydrin.
 It is a colorless liquid with a pungent, garlic-like odor, moderately soluble in water,
but miscible with most polar organic solvents.
 what is protein precipitation
• Is a methods
• are used for the concentration of diluted proteins in solution.
• The goal is to purify and concentrate contaminated proteins or proteins contained
in various matrices such as buffers, detergents or in natural sources, such as blood,
urine or other biofluids.
• Protein precipitation methods are routinely used to concentrate and eliminate
interfering molecules prior to electrophoresis or quantitative protein
characterization or determinations.
• The mechanism of protein precipitation is to alter the solvation potential of the
solvent.
• The solubility of the solute is lowered by adding a specific reagent to manipulate
the repulsive electrostatic forces between proteins to favor their aggregation.
• The solubility of proteins in aqueous buffers depends on the distribution
of hydrophilic and hydrophobic amino acid residues on the protein's surface.
• Hydrophobic residues predominantly occur in the globular protein core, but some
exist in patches on the surface.
• Proteins that have high hydrophobic amino acid content on the surface have low
solubility in an aqueous solvent.

• Charged and polar surface residues interact with ionic groups in the solvent and
increase the solubility of a protein. Knowledge of a protein's amino acid composition
will aid in determining an ideal precipitation solvent and methods.

• Protein precipitate formation occurs in a stepwise process.

• First, a precipitating agent is added and the solution is steadily mixed. Mixing causes
the precipitant and protein to collide.

• Salting out is the most common method used to precipitate a protein.

• Addition of a neutral salt, such as ammonium sulfate, compresses the solvation layer
and increases protein–protein interactions. As the salt concentration of a solution is
increased, the charges on the surface of the protein interact with the salt, not the
water, thereby exposing hydrophobic patches on the protein surface and causing the
protein to fall out of solution (aggregate and precipitate).
 Y ammonium sulfate using in protein
precipitation?
• Ammonium sulfate precipitation is one of the
most commonly used methods for large and
laboratory scale protein purification and
fractionation that can be used to
separate proteins by altering their solubility in
the presence of a high salt concentration. ...
This can be seen with the folding of
recombinant proteins.
What is the use of salting out process?
• The process of "salting out" is a purification
method that relies on the basis of protein
solubility. It relies on the principle that most
proteins are less soluble in solutions of high
salt concentrations because the addition of
salt ions shield proteins with multi-ion
charges.
How does salt precipitate the protein?
• However, when the salt concentration increases, the
solubility decreases, and with certain salts
proteins become insoluble (salting-out). The
concentration must be very high to precipitate
proteins; therefore very soluble salts are generally
used for protein precipitation (Hofmeister series).

• Adding of an appropriate percentage of salt to a

solution causes the water molecules to be exhausted
gradually from the surrounding of the desired
molecules that are polar in the water.
 How does solubility affect the
precipitation?
• Precipitation reactions occur when cations
and anions in aqueous solution combine to
form an insoluble ionic solid called
a precipitate.
• Whether or not such a reaction occurs can be
determined by using the solubility rules for
common ionic solids.
 What is IEP precipitation?
• The precipitation of a protein at
its isoelectric point, at which proteins are
generally least soluble. It is useful in the
fractionation of mixtures of proteins.
 Why protein less soluble in IEP?
• It is positively charged at low pH and
negatively charged at high pH. The
intermediate pH at which a protein molecule
has a net charge of zero is called theisoelectric
point of that protein. ... As a result, protein is
the least soluble when the pH of the solution
is at its isoelectric point.
 What is protein solubility?
• This pH is called the isoelectric point, and for
most proteins it occurs in the pH range of 5.5
to 8. A protein has its lowest solubility at its
isoelectric point.
• If there is a charge at the protein surface,
the protein prefers to interact with water,
rather than with other protein molecules.
 Does salt denature the protein?
• Heavy metal salts act to denature proteins in
much the same manner as acids and bases.
• Heavy metal salts usually contain Hg+2, Pb+2,
Ag+1 Tl+1, Cd+2 and other metals with high
atomic weights.
• Since salts are ionic they disrupt salt bridges
inproteins.
 Fractional precipitation
• Fractional precipitation is a technique
• that separates ions from solution
• based on their different solubilities.
• For example, consider an aqueous solution that contains initially Ba2+ and
Sr2+ ions.
• a method of separating a mixture of substances by means of their gradual
precipitation from a solution.
• The possibility ofquantitative separation of a mixture depends on the ratio
of the original concentrations of compounds being precipitated andon the
values of their solubility product.
• Coprecipitation of a more soluble compound with a less soluble one is pos
sible in theprocess of fractional precipitation.
• The methods of fractional precipitation, which are generally used for separ
ation of mixturesof substances that are close in terms of chemical properti
es and solubility, are extremely labor-intensive.
 Fractional precipitation
• is a technique that separates ions from solution based on their
different solubilities. For example, consider an aqueous solution
that contains initially Ba2+ and Sr2+ ions. Suppose that an aqueous
solution of potassium chromate (K2CrO4) is added slowly. Two
precipitates can form: BaCrO4 with Ksp of 1.2 x 10-9 M, and
SrCrO4 with Ksp of 3.5 x 10-5 M. (Since all potassium salts are
soluble, the potassium ion of the aqueous solution of K2CrO4 is a
spectator ion and does not need to be considered in the solubility
equilibrium.) Because Ksp for SrCrO4 is smaller than for BaCrO4,
SrCrO4 will precipitate first. If K2CrO4 is added to the solution so that
the ion product for BaCrO4 is not exceeded, then only SrCrO4 will
precipitate. The resulting solution can be filtered and the
Sr2+ isolated from the original solution as the chromate salt.
 elution
• is the process of extracting one material from another by washing
with a solvent; as in washing of loaded ion-exchange resins to
remove captured ions.
• to the chemical process of altering a material by stripping its ions
via an ion exchange with another material. It is a vital process to
prevent corrosion by facilitating augmentation and inhibition of
corrosion by exchanging corrosion prone ions with more resistant
ones on a substrate metal.
 dialysis
• a semipermeable membrane is used to separate small molecules
and protein based upon their size.
• The bag is filled with a concentrated solution containing proteins.
• Molecules that are small enough to pass through the pores of the
membrane diffuse out of the bag into the buffer solution, or
dialysate.
• diàlysis, "dissolution";
• dià, "through", a
• λύσις, lỳsis, "loosening or splitting")
How long?
Load the sample into dialysis tubing, cassette or device and
dialyze for 2 hours. You can perform this step at room temperature
or 4°C. Change the dialysis buffer and dialyze for another 2 hours
 How much protein dialysis per day?
• For those on dialysis, the recommendation is 0.55
grams of dietary protein perpound of body weight.
• This is higher than the 0.36 grams recommended for
the average healthy individual and, of course, higher
than the 0.25 grams per pound of body weight used
with the pre-dialysis diet.

• dialysis membranes made of polysulfone,

polyethersulfone (PES), etched polycarbonate, or
collagen are also extensively used for specific medical,
food, or water treatment applications.
 Protease vs proteinase
• protease are enzyme that degrade proteins by
hydrolysis of the peptide bond . but some of
the proteolytic enzymes act best on intact
protein.
• proteinase are the protease that show
specificity for the intact protein.
 disulfide bond
• , also called an S-S bond,
• disulfide bridge,
• is a covalent bond
• derived from two thiol groups.(sulfer)
• In biochemistry, the terminology R-S-S-R connectivity is commonly used to
describe the overall linkages. The most common way of creating
this bond is by the oxidation of sulfhydryl groups.
• can be broken by addition of reducing agents.
• this bond is creating by the oxidation of sulfhydryl groups.

to stabilize the tertiary and/or quaternary structures of proteins and may be

intra-protein (i.e., stabilizing the folding of a single polypeptide chain) or inter-
protein (i.e., multi-subunit proteins such as antibodies or the A and B chains of
insulin). they are very important in determining the quartenary structure of
some proteins. An very prominant example would be the role ofdisulfide
bonds in the structure of antibody molecules.
Hairs are made of keratin molecules, which contain cysteine. Cysteine has thiol
(-SH) group, by which it can form disulfide (-S-S-) bond with another cysteine
of another keratin, causing bending of hair.
 DIGE
• Difference gel electrophoresis (DIGE)
• is a form of gel electrophoresis
• where up to three different protein samples can be labeled with size-
matched, charge-matched spectrally resolvable fluorescent dyes (for
example Cy3, Cy5, Cy2) prior to two dimensional gel electrophoresis.
• s a modification of 2D-PAGE that requires only a single gel to reproducibly
detect differences between two protein samples.

PRINCIPLE:
Two different protein mixtures are labeled with two different fluorophores, 1-(5-
carboxypentyl)-1′-propylindocarbocyanine halide (Cy3) N-hydroxysuccinimidyl ester and
1-(5-carboxypentyl)-1′-methylindodicarbocyanine halide (Cy5) N-hydroxysuccinimidyl
ester fluorescent dyes, respectively. The labeled proteins are mixed and separated in the
same 2D gel, thus enabling the analyst to run two different samples on the same gel in a
2D format
 cy3
• Cyanine
• is the non-systematic name of a synthetic dye family
• belonging to polymethine group.
• The word cyanin is from the English word "cyan", which conventionally
means a shade of blue-green (close to "aqua")
• is derived from the Greek κυάνεος/κυανοῦς kyaneos/kyanous which
means a somewhat different color: "dark blue".
• Cyanines were and are still used in industry,
• more recently in biotechnology (labeling, analysis).
• Cyanines have many uses as fluorescent dyes,
• particularly in biomedical imaging.
• Depending on the structure, they cover the electromagnetic
spectrum from near IR to UV. There are a large number reported in the
literature.