I have a feed containing B2M, albumin and antibody fragments from which i want to purify B2M as my sole target protein using Chromatography resin in one or two steps.
If anyone has any good suggestions on the same, please help.
The method for purification of b2m from albumin would depend on factors such as the starting amounts of each, whether you need to retain all of the b2m or just end up with some pure b2m and the presence of other molecules and also the starting amounts of the crude product. With only a small amount of starting material you could use specific binding ( antibody/binding proteins) columns or isoelectric focussing. If starting with large amounts of material size exclusion sephadex columns ( b2m is 12000 D and albumin is 66.5kd so sephadex g75 would separate most of the albumin leaving a purer b2m for further purification by ion exchange chromatography or IEF ( as the pi of the proteins are very different)
Use a spin column with 20 kDa cut off and use filterate for purification of microglobulin on a Sephadex G-50 column. You will get purified b2m.
Shamsher sir, I tried the method suggested by you, excluding the Sephadex G50 run (which i would try the same as soon as i receive this gel). Initial results from the SDS PAGE analysis shows presence of B2M protein in the permeate, which i loaded on DEAE column, still full separation of B2M from the contaminants has not been full achievable. I agree that, GFC is required to have a fully purified protein in this case, but i want to try it out using a single step of IEX after 20kD separation. Can you (or anyone) please suggest any good weak ion exchanger other than DEAE, would be helpful to try to out.....:)
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