Hello Attiya,

Indeed, SDS-PAGE should not be able to resolve Mw differences caused by point mutation of a single amino acid. The differences of migration you see are due to a change of conformation of your protein or to a different ability to bind SDS. Nevertheless, it is rather strange that it should occur owing to a single mutation, particularly with the valine to isoleucine mutation. Alternatively, it could be that the preps you analyze undergo different patterns of proteolysis. Mass spectrometry may help in clarifying the question;

Thanks Pierre for your message. What do you mean by particularly with the valine to isoleucine? i.e. the overall charge is the same?

Sometimes a single change can cause the molecule to fold differently as Pierre says so many amino acids which were buried internally in the molecule may now find themselves on the surface of the protein causing quite a large change in the external charge on the protein so a large effect on the mobility of the protein in an electric field

Thank you Paul for your reply. Would changes in post-translational modifications cause an possible effect in migration?

Hi Attiya, I have also observed similar changes in migration pattern following point mutations. As others suggested it has to do something with the change in conformation. I don't know if you are trying to purify the proteins, then you can see if that is indeed the case by using techniques like SAXS or DLS. However, the residues you mention are usually not involved in any post-translational modifications. Nonetheless, you can check the surrounding residues for any known/predicted post-translational modification of your protein which might be affected by your mutations.

Thank you for your reply Feroj. Your suggestions are really helpful. PTMs haven't been studied much in my protein of interest but I suppose I can look into predicted sites.