Hi Rongjin, the concentration (and total amount) of detergent during purification depends a lot on what stage of purification you're at. For membrane extraction (1st step), people typically use anything from 1-5%. This depends on your budget, on the cmc of the detergent, and of course on the amount of cells you have. b-OG, having a high cmc (~0.7%) is not very effective at 1%, so you have to use a higher concentration. 1% is often enough for things like DDM and LDAO. My lab uses typically 100 ml of 1% detergent to extract membrane proteins from 12 l Ecoli culture (so 1 g detergent total). Later on in purification, when the amounts of stabilizing endogenous lipids decrease, it makes sense to lower the detergent concentration as too high concentrations are often denaturing. I would not go lower than ~ 2-fold cmc (it is hard to know what the actual cmc is in your buffer). In the case of DDM, we do our 1st purification step in 0.2% DDM (Ni-column) and subsequent steps in 0.03-0.05% DDM (approx. 2x cmc). 2% DDM for purification seems excessive and will very likely not allow crystallization (if you're attempting that). Purification in one detergent followed by exchange later is perfectly fine. However, it seems your protein is not very happy in DDM, so I think it would make sense to start off (extraction) from a different detergent. Cheers, Bert Bert van den Berg University of Massachusetts Medical School Program in Molecular Medicine Biotech II, 373 Plantation Street, Suite 115 Worcester MA 01605 Phone: 508 856 1201 (office); 508 856 1211 (lab) e-mail: [EMAIL PROTECTED] http://www.umassmed.edu/pmm/faculty/vandenberg.cfm
________________________________ From: CCP4 bulletin board on behalf of Rongjin Guan Sent: Fri 12/21/2007 2:09 PM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] detergent concentration used for membrane protein purification Dear All sorry for a non-ccp4 related question. what is the typical concentration of detergents used in membrane protein purification? Is it usually within 1-10 times of CMC? My collaborator was using 10% DDM in purifying a membrane protein. I suggested to reduce it and now he is working with 2% DDM. He said lower than that the protein is not soluble. so another question is, can we purify the membrane protein first at high concentration of one detergent (say, 2% DDM), and then screen different detergents later to find a better one? Or it is better to screen the detergents first before purification? any suggestions will be appreciated. Have a nice holiday! Rongjin Guan
