Raji, First, I would suggest increasing the solubilization time by attempting solubilization overnight at 4 degrees. I would also suggest a few more detergent choices. In addition to DDM, my other favories are OG and Elugent (a mixture of several detergents) for extraction from *E. coli* membranes. If one of these still doesn't extract your protein of interest, you can move onto other more exotic options. I know it's a bit self-plugging, but the products division of Emerald Bio sells a 96-well detergent screen you can use to go through a wide variety of solubilization detergent options at small scale. We've used it in-house in our services division to get some nice results for several of our membrane protein projects.
However, you may be fighting a losing battle as many times protein that doesn't extract from the membranes can be misfolded or aggregated, remaining strongly associated with the membranes. Cheers, Jim On Wed, Jul 10, 2013 at 2:23 PM, Raji Edayathumangalam <r...@brandeis.edu>wrote: > Dear BBers, > > Sorry for the non-ccp4 post. > > I'd like to hear tips and suggestions from the membrane protein folks in > the community. > > I am currently purifying two different membrane proteins expressed in E. > coli. While I am able to extract practically all of my first protein from > the cell membrane into buffer containing 1% DDM in 1hr at 4C, it doesn't > seem as though that works very well for my second protein. I understand > every protein is a unique beast; I am just trying to increase the yields > for my second protein as much as possible. > > For my second protein, I have tried solubilizing for 1hr at 4C in 1% DDM > as well as in 1-2% NM for 1hr at 4C but am only able to solubilize about > half of total protein in the membrane. Have folks seen substantial increase > in % solubility with longer incubations with detergent? Or should I > consider the issue that the fraction that doesn't solubilize may be > misfolded, just cut my losses and grow tons more bacterial cultures. > > Many thanks for sharing your successes and heartaches on this matter! > Raji > > -- > Raji Edayathumangalam > Instructor in Neurology, Harvard Medical School > Research Associate, Brigham and Women's Hospital > Visiting Research Scholar, Brandeis University > > -- Jim Fairman, Ph D. Crystal Core Leader I Emerald BioStructures <http://www.emeraldbiostructures.com/> Tel: 206-780-8914 Cell: 240-479-6575 E-mail: fairman....@gmail.com jfair...@embios.com
