Question

Gel electrophoresis. Polypeptides can be separated by electrophoresis according to their relative content of acidic and basic residues. The isoelectric point (pI) of a polypeptide is the pH at which its net charge is zero. (See Fig 3.11 of your textbook). A) At physiological pH (= 7.4), what is the net charge on the tripeptide Asp-Arg-Glu-His? B) Calculate the pI of this tetrapeptide. [Hint: Using the pK, values in Table 2.1 and the Henderson-Hasselbalch equation, determine the pH when the population of tripeptides in aqueous solution have an average charge = 0.] C) Briefly explain the principle of SDS-polyacrylamide gel electrophoresis. What is the purpose of the SDS? Which proteins midrate slowest by SDS-PAGE? D) Briefly explain Western blotting. How are proteins detected? Sketch the polypeptide structure of an antibody used in Western blotting. Where does an antigen bind to the antibody?

Answer 1

A) pI of the peptide Asp-Arg-Glu-His is 5.32 charge is zero

generally below the PI the protein is Positively charged and above the pI the charge is negative.

so at pH 7.4 the chage is -1

(CH2)s 13 12.5 (CH2) 10.5 HN (CH2H (CH2)a COO H2 COOH COOH CH2 CH2 CH2 histidine pK-6.0 aspartic glutamic lysine arginine pK-10.5 pK-12.5 acid acid pK-4.2 pK-3.9

B) at the pH 5.32 the protein has zero charge (table is not available) calculated from bioinformatic tools

c) high molecular weight proteins migrate slowly in SDS page

PAGE
Polyacrylamide gel electrophoresis (PAGE) is probably the most common analytical technique used to separate and characterize proteins. A solution of acrylamide and bisacrylamide is polymerized. Acrylamide alone forms linear polymers. The bisacrylamide introduces crosslinks between polyacrylamide chains. The 'pore size' is determined by the ratio of acrylamide to bisacrylamide, and by the concentration of acrylamide. A high ratio of bisacrylamide to acrylamide and a high acrylamide concentration cause low electrophoretic mobility. Polymerization of acrylamide and bisacrylamide monomers is induced by ammonium persulfate (APS), which spontaneously decomposes to form free radicals. TEMED, a free radical stabilizer, is generally included to promote polymerization.

SDS PAGE
Sodium dodecyl sulfate (SDS) is an amphipathic detergent. It has an anionic headgroup and a lipophilic tail. It binds non-covalently to proteins, with a stoichiometry of around one SDS molecule per two amino acids. SDS causes proteins to denature and dissassociate from each other (excluding covalent cross-linking). It also confers negative charge. In the presence of SDS, the intrinsic charge of a protein is masked. During SDS PAGE, all proteins migrate toward the anode (the positively charged electrode). SDS-treated proteins have very similar charge-to-mass ratios, and similar shapes. During PAGE, the rate of migration of SDS-treated proteins is effectively determined by molecular weight.

SDS is a detergent that is present in the SDS-PAGE sample buffer where, along with a bit of boiling, and a reducing agent (normally DTT or B-ME to break down protein-protein disulphide bonds), it disrupts the tertiary structure of proteins. This brings the folded proteins down to linear molecules.

SDS also coats the protein with a uniform negative charge, which masks the intrinsic charges on the R-groups. SDS binds fairly uniformly to the linear proteins (around 1.4g SDS/ 1g protein), meaning that the charge of the protein is now approximately proportional to its molecular weight.

D)Western blotting, also known as immunoblotting or protein blotting, is a core technique in cell and molecular biology. In most basic terms, it is used to detect the presence of a specific protein in a complex mixture extracted from cells. The Western blotting procedure relies upon three key elements to accomplish this task:

the separation of protein mixtures by size using gel electrophoresis;

the efficient transfer of separated proteins to a solid support;

and the specific detection of a target protein by appropriately matched antibodies.

$WBQ_home-Fig1.jpg$

antigen binds at hyper variable regions of antibodies which are specific to it

epitopes of antigens are bound to paratopes with in antibodies

structure of antibody has 2 light chains and two heavy chains

simply it can be notated as bellow

antigen- binding site variable region light chain heavy chain constant region Schematic design of an Immunoglobulin (lgG)