Hi Vijay, It may be caused by the redox status of your proteins, which is normal in redox related proteins, especially caused by cysteine residues.
The upper band may be the reduced form and the lower oxidized form, which can be found in numerous papers. Addition of oxidants or reductants in purification may solve the question and homogenize your proteins, regardless of the result of SDS-PAGE. First of all, check the redox status of your proteins. Best Regards and Good Luck! Sincerely yours, Jiang Yu School of Life Sciences University of Science and Technology of China _____ 发件人: CCP4 bulletin board [mailto:[EMAIL PROTECTED] 代表 Vijay Kumar 发送时间: 2007年11月4日 20:22 收件人: CCP4BB@JISCMAIL.AC.UK 主题: [ccp4bb] protein degradation? 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
