Thanks Jim and Bert for all your suggestions. I plan to increase time and/or amount of DDM and see what happens before switching detergents. I'll keep the suggestions about other detergents and 96-detergent screen in the back of my mind.
Bert, I noted your suggestion of also using a larger amount of total detergent and will definitely try that. Jim, thanks for suggesting Elugent. Never heard about it before so great to know. Many thanks and cheers, Raji On Wed, Jul 10, 2013 at 5:23 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 > > -- Raji Edayathumangalam Instructor in Neurology, Harvard Medical School Research Associate, Brigham and Women's Hospital Visiting Research Scholar, Brandeis University
