Hi, I have been trying purify a N-ter his-tagged protein over-expressed in E.coli. After purification (Ni-NTA or Co-Talon), I find two bands in SDS PAGE which are very close each other (top band in the right MW and more intense than the lower band). Western blot (for his-tag) of the gel gave signal for both the bands. Mass spec results confirmed both protein bands are the same. So I think it could be C-ter degradation of my protein. Also the 2 bands exist after ion-exchange and sizing column.
I use commercially available complete protease inhibitor tablets (increasing concentration has no effect) and sonication for lysis. I am wondering if people have encountered the same problem and got any suggestions? Thanks in advance. Regards, Vijay
