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Introduction

Separation of protein is carried out using cross-linked polyacrylamide gel electrophoresis. This is due to molecular sieving property of polyacrylamide gel electrophoresis that slows the migration of protein according to their molecular weight. Proteins were originally separated on polyacrylamide gels that were polymerized in a glass tubes, approximately 7 mm in diameter and about 10cm in length. SDS- polyacrylamide gel electrophoresis is the most widely used method for analysis of protein mixtures qualitatively. Polyacrylamide gel electrophoresis is used for monitoring protein purification. As the separation of protein is according to size the method can also be used to determine the relative molecular mass of protein.

Principle

SDS an anionic detergent is used for separating proteins by masking the original charge of the protein molecule so that net charge of the protein is positive. Charge of SDS and hence they move towards the anode. The rate of migration depends on the molecular weight of the protein.

Materials required

Gel: Cross-linking monomers of acrylamide and bisacrylamide by polymerization in presence of catalyst TEMED and chain initiator ammonium per sulphate. Polymerization of acrylamide and bisacrylamide can also be carried out using light in presence of riboflavin. This type of polymerization is called photo polymerization.

SDS: Sodium dodecyl sulphate

It is an anionic detergent, which binds to the protein and masks its charge. It binds to the polypeptide in a ratio of one SDS for every two amino acid residue.

Beta mercapto ethanol:

It breaks the disulphide bridges. These disulphide bonds normally are responsible for 3d structure of protein.

Tris buffer of different pH: Tris glycinate buffer pH 8.3
Tris Hcl buffer pH 6.6

Sucrose or glycerol:

They increase the density of the sample so that once the sample is loaded it just sinks into the well.

Bromophenol blue:

This is called tracking dye and this dye is used to monitor electrophoretic run.

CBB-R250: coomassive brilliant blue

Most commonly used stain for detecting protein and it is a sulphated tri methylamine dye.

Initial procedure

Proteins are treated with beta mercapto ethanol, which cleaves the disulphide linkage. This protein is then treated with SDS when they get denatured.

Detection and estimation

Non-fluorescent molecule can be allowed to flourecine. Fluoresamine dye bind to primary amine groups of lysine to form adduct which fluoresce under UV radiation. If the sample is radiolabelled then the protein can be detected by autoradiography.