Question

R5-1. An antibody specific for a protein of interest can be utilized in which of the following protein separation techniques.

a.     Ion-exchange chromatography

b.    affinity chromatography

c. western blot

d.    Edman degradation

e.     A and B

f.      B and C

Answer 1

Ans) F. B & C (affinity chromatography & western blot)

Affinity chromatography: the main principle of this technique is affinity. The natural affinities between the two compounds are utilized for the separation of molecules.

Example for the pairs of compounds that show natural affinities

Antigen and antibody, Hormone and receptor, Enzyme and substrate, Ligand and receptor

In Affinity chromatography to separate a desired protein, the antibody against the desired protein is raised which specifically recognizes protein. The antibody attached matrix packed into the column and the protein sample is passed through the column. Due to the affinity, the matrix connected antibody attaches to the desired protein and remaining proteins are eluted out. Later with the help of specific buffers, our desired protein is eluted out.

Western blot: it is also referred to as immune blot and used to separate the protein of interest from a tissue sample or homogenate mixture of the sample with the help of antibody specific to the protein of interest. In Western blot first the proteins are immobilized on a membrane and then antibody (primary) is added. The presence of protein detected by the use of the enzyme-linked secondary antibody.