Hi Raji, the amount of detergent used is more important than the concentration. The more cells you have, the more detergent is required. To give you an idea, we tend to use 100 ml of 1% detergent (=1 g) for 12 liters of cells (Ecoli) of OD600 1. This is probably on the low side; if cost weren't an issue I'd prefer using at least 2% (2 g) for this amount of cells. So, the lower extraction yield for your second protein could simply be due to higher ODs. To increase yields you can either (as Jim said) increase extraction time and/or increase the amount of detergent. Also, the milder the detergent (like DDM), the less efficient it tends to be in extraction. So, 1 g of DM will be more efficient than 1 g of DDM, but of course the DM may not keep your protein happy.....For very stable proteins, the detergents to use are LDAO and Elugent (a mixture of alkylglucosides); these are also very cheap compared to DDM. A final possibility is indeed that some of the protein may be misfolded and "unsolubilizable".
Good luck, 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
