Just a thought—is the reducing agent changing pH? Also, is your protein a metal-binder?
JPK From: CCP4 bulletin board [mailto:[email protected]] On Behalf Of zhuqing ouyang Sent: Monday, May 12, 2014 12:33 PM To: [email protected] Subject: [ccp4bb] off topic, complex precipitate in reducing reagents Hi all, I came across a strange problem. When I purify a protein complex, let's say AB, it will precipitate when I add reducing reagent like DTT or TCEP in the gel filtration buffer, or if I don't include any reducing reagent running column, I take the peak fractions and supplemented with DTT or TCEP, it will also precipitate. And the precipitation is irreversible when I change buffer to non-reducing reagent buffer. But when I purify A or B alone, they don't have this problem. Anyone have some clues for this observation? Thanks a lot. zhuqing
