Dear friends,
I have two problems with my proteins. For protein A, it was purified using the Nickel-affinity purification method. If the sample was placed at 4 degrees centigrade for 24 hrs, it didn't change obviously. But if it was concentrated by centrifuge at 4 degrees for further gel-filtration purification, it degraded very heavily during the concentrating process (~4hr), resulting there is nearly NO band corresponding to the full-length protein B nor any band corresponding to fragment of protein B. So I think protein B may undergo nonspecific degradation into very small fragments. I have used PMSF, Leupeptin, Pepstantin, and Aprotinin as protease inhibitor mixture to preventing the degradation, but it didn't work. For protein B, it can be purified to the purity of >95%, and can be concentrated to >20 mg/ml (although a little slowly). When setup hanging-drop crystallization trials, it was found phased heavily under most conditions. I have tried different buffer systems (Tris-HCl or NaH2PO4), with or without cofactor or substrate analogue. But none of these worked. The protein still phased very heavily. Would somebody share their experience on preventing protein degradation during purification or preventing phasing during crystallization. Any comments or suggestion are welcome. Thanks in advance. Yingjie Yingjie PENG, Ph.D. student Structural Biology Group Shanghai Institute of Biochemistry and Cell Biology (SIBCB) Shanghai Institute of Biological Sciences (SIBS) Chinese Academy of Sciences (CAS) 320 Yue Yang Road, Shanghai 200031 P. R. China 86-21-54921117 Email: [EMAIL PROTECTED]
