Question

How would you combine affinity chromotography together with size exclusion chromatography to improve separation?

In the equilibrate step of the affinity chromotography protocol, you accidentally use the elution buffer instead of the affinity binding buffer. How will this affect the purification process?

Answer 1

Affinity chromatography is highly selective for the separation of specific proteins. Ligand of specific group is binded to the solid phase, which binds to types of proteins having legand groups, these binded proteins some times may be more than 1 types but has different molecular size. So to again purify this proteins size exclusion chromatography is applied. In affinity chromatography unbound proteins removed by using equalibration buffer after that bind proteins are eluted by using eluting buffer which elute proteins of interest, this eluted sample, load on the size exclusion chromatography directly which elute the proteins on the basis of their sizes, this highly improve the separation.

If in affinity chromatography we accidentally used elution buffer instead of affinity binding buffer then no separation occur, because affinity binding buffer does not affect the binding of ligand binded proteins, it only removed unbound proteins and doesn't remove binded proteins this way it removes unwanted proteins from proteins of interest. After that column eluted with elution buffer which has ability to remove ligand binded proteins from ligand. This cause separation of samples. If we directly used elution buffer then no separation occure.