Dear Teresa, In addition to Bert's excellent list, I have found that many membrane proteins aggregate in regular SDS-PAGE sample buffer, particularly when heated. In the best case, we always see "dimers" or, in the worst case, a ladder or smear of aggregates. If you heat your SDS-PAGE samples, try not heating them. Secondly, your observation that you have protein eluting near the void volume on Superdex 200 may also suggests that you have either incomplete solubilization (i.e., big lipid-protein complexes) or, as Bert remarks, an unhappy protein that aggregates in the detergent you have chosen.
However, before you consider this a problem, ask yourself if this is an expected result due to the heterologous overexpression of a recombinant protein. Unlike purifying a "native" protein, overexpression of a recombinant protein can led to a considerable amount of misfolded or misassembled protein mixed with good protein. As you might be overloading the membrane protein insertion system, you may need to dial back the induction. Finally, were you measuring the amount of high order aggregates in SEC by A280 or by another means? The proportion of high order aggregates observed near the void volume is always skewed due to light-scattering of the much larger particles. Because of this, a large void volume peak (~7.5-8 mL on a Superdex 200 10/300 column) may contain a lot less protein than you think. Good luck, Michael **************************************************************** R. Michael Garavito, Ph.D. Professor of Biochemistry & Molecular Biology 603 Wilson Rd., Rm. 513 Michigan State University East Lansing, MI 48824-1319 Office: (517) 355-9724 Lab: (517) 353-9125 FAX: (517) 353-9334 Email: [email protected] **************************************************************** On Jul 9, 2013, at 4:47 AM, Bert Van-Den-Berg wrote: > 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
