Question

Explain one other method (besides electrophoresis) that could be used to purify proteins and compare it to Nickel IMAC?

P.S. Need to explain one method to separate and purify proteins but not electrophoresis, as we have done that in class.

Answer 1

Chromatography technique of protein purification-

The basic procedure in chromatography includes flow of the protein solution through a column packed with various materials. This function on the principle that different proteins interact differently with the column material, and can thus be separated by the time required to pass the column. In this technique generally proteins are detected as they are coming off the column by their absorbance at 280 nm.

Immuno-affinity Chromatography:

This technique uses the specific binding of an antibody to the target protein for the purpose of selectively purifying the protein. The procedure involves immobilizing an antibody to a column material, which then selectively binds the protein, while everything else flows through.

Nickel IMAC technique-

Nickel columns are used for immobilized metal affinity chromatography for the purification of recombinant protein with a polyhistidine tag on any one of the terminus. Nickel gives highest yield because a recombinant protein with a 6xHis tag has high affinity for nickel. But most other protein will bind with low affinity or not bind at all.