I could add that even for membrane proteins that may not be stable in eg DM after purification, DM may still be a viable option for extraction since there will be large amounts of stabilising lipids present. Just make sure you change to a milder detergent for the post-extraction steps.
Bert ________________________________ From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Raji Edayathumangalam [r...@brandeis.edu] Sent: Wednesday, July 10, 2013 10:23 PM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Detergent solubilization step (membrane proteins) 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
