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

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