Question

1) You are faced with separating two different proteins with identical mass. Protein A is a rod-like molecule with a great deal of secondary srtucture and protein B is a globular protein that is roughly spherical. What methods could be used to separate these two proteins? (check all that apply).

A. Ion-exchange chromatography

B. Two-dimensional electrophoresis

C. SDS PAGE

D. Size exclusion chromatography

E. Affinity chromatography

Answer 1

Answer is A, B and E. we can use Ion exchange chromatography, Two dimensional electrophoresis, Affinity chromatography to separate the above proteins.

We can’t use SDS PAGE and size exclusion chromatography as both separates the proteins based on their size. You can see the explanation below.

Sizeexclusion chromatography	separates proteins based on size, larger proteins will elute faster than smaller ones
Ion-exchange chromatography	Separation is based on differences in the sign and magnitude of the net electric charge of proteins at a given pH. Two types of matrix. cation exchangers- synthetic polymer with bound anionic groups anion exchangers- synthetic polymer with bound cationic groups
Affinity chromatography	Based on binding affinity and beads in the column with have covalently attached ligand and the protein with affinity towards towards this ligand will go and bind to this and migration is retarded, thus separation is achieved.
SDS PAGE	Separates based on molecular weight of the protein. SDS unfolds the protein and will bind to it, thus gives negative charge. Thus polypeptides migrate on SDS PAGE, smaller proteins migrate faster.
Two dimensional electrophoresis	Combination of isoelectric focusing and SDS electrophoresis. Separates proteins of identical molecular weight that differ in pI, or proteins with similar pI values but different molecular weights.