You are seeing proteins at 30, 40, 50 and 70 kDa because you are probably in denaturing conditions (SDS PAGE ?). When you concentrate your samples you have interactions that cause the proteins not to be in the cutoff 100kDa flowtrought.

Maybe you should add an extra step of Size Exclusion Chromatography.

Dear Abhinav Hatwar

There are several reason why in general the ultrafiltration differently to SEC is not so good to separate protein with lower MW respect than higher MW.

As Nicolas Poirier is possible that the proteins are not monomeric and therefore their MW do not correspond really to the MW observed in SDS-page but there are also at least 2 other reasons;:

1)Differently to SEC, where you are diluting your sample during the run, in ultrafiltration you have a concentration effect that my induce aspecific intra molecolar interaction can be more strong and the proteins may interact and form aggregates

2) The MW cut-off is defined considering the protein as a sort of sphere but is it possible that some proteins as also quite different shape and hydrodinamic radius that can therefore not be in agreement with their MW

I tihnk that ultrafiltration is a good way to concentrate the proteins and some times even to do rude buffer exchange but not for protein purification.

best

Manuele

Hello Abhinav, more details would help us troubleshoot. What protein are you studying and what is the molecular weight? What is buffer composition of your buffer for binding to the resin during metal-affinity chromatography? Can you kindly provide a PDF of your protocol and images of the polyacrylamide gel? It sounds like your protein is quite large and things may be sticking to it. That would also explain why the lower molecular weight species below the Amicon filter size cutoff persist following concentration. If so, increasing the salt concentration in your buffer may help prevent non-specific interactions. If need be, you can immobilize your protein on the resin and try to run NaCl and pH gradients to see if you can disrupt those non-specific interactions. Another possibility, though less likely, is that those lower molecular weight species are ”not real” and they are merely breakdown products as a result of you boiling your sample in SDS-PAGE dye (this has happened to me before)— how long do you boil your samples for? Thanks!

I completely agree with the points mentioned above. Ultrafiltration is primarily for concentration, not purification. As discussed, protein structures do not have an ideal shape. Due to protein association, they may exist as multimers rather than monomers, resulting in a much larger actual solution size than expected.

In the amicon cell you will have high concentration of proteins close to the membrane. Sometimes that can cause build up of a gel layer on the membrane. Once that happens this gel layer is determining the cutoff - not the membrane.