since your protein aggregates even in a mild detergent you may have to find an ortholog that is more stable. however, there are a few things you can try before moving on (in arbitrary order): 1. add glycerol during purification (5-20%) 2. get rid of the imidazole as fast as possible after Ni. Many proteins do not like imidazole at all. 3. explore different buffer conditions during purification, especially regarding ionic strength and pH. 4. adding reducing agents may help, but given that your aggregates are large I expect this may not help that much. 5. add lipids and/or substrate/inhibitor etc during purification.
Good luck, Bert ________________________________________ From: CCP4 bulletin board [[email protected]] on behalf of Theresa Hsu [[email protected]] Sent: Tuesday, July 09, 2013 5:01 AM To: [email protected] Subject: [ccp4bb] Heterogeneity during purification Dear all I am working on a 30 kDa membrane protein which forms a functional dimer. The protein is His-tagged at N-terminal. In small scale expression screening from whole cells, there is only a single band on Western blot at 30 kDa. But, after purification, additional bands appear at 60 and 120 kDa on SDS-PAGE and Western blot. On size exclusion with Superdex 200, a large proportion elute near the void volume (8 ml). Detail purification For small scale screening, I lysed cells in 20 mM Tris pH 8, 100 mM NaCl, 1 mg/ml lysozyme, 1 % DDM and DNAse for 2 hours and then centrifuged at 16000 g. I then checked the supernatant on SDS-PAGE and scale it up for purification. For purification, I use the buffer 50 mM Tris pH 8, 300 mM NaCl, 20 mM imidazole, 0.05 % DDM (two times CMC of DDM). Is there suggestion to get homogeneous protein? Thank you. Theresa
