Hi Rongjin, During extraction from the membrane, typically 10xCMC (e.g. FC12) to 100xCMC (e.g. DDM) may be used, depending on target under study. During purification, it is recommended to bring this down to ~2xCMC. Theoretically, 1xCMC should be ok to keep the protein soluble. In practice, it is difficult to get exactly 1xCMC in solution as the CMC value will fluctuate depending on salt in the buffer, solution temperature, inaccuracies in mass measurement when making the detergent solution, etc. Hence it is preferable to stay ~2xCMC. If you then use a 100K concentrator post purification, you should be fine. I have had a couple of cases when the protein seemed unstable at what we thought as 1xCMC but was ok at 2xCMC. Yes, you can purify the protein first at a high conc of one detergent, and then try detergent exchange during a SEC step into different detergents prior to crystallization. The problem with this is that detergent exchange is dependent on CMC value. If CMC value is very low, it is difficult to do a detergent exchange since micelles will form even at very low concentrations which may be difficult to get rid of. So, if you want to go this route, better to keep in mind the CMC value and proceed accordingly. It would also be ok to screen detergents at the extraction step and find the one that solubilizes/stabilizes best and then keep the same detergent throughout until crystallization. Since so much remains unknown about membrane proteins and best detergents, it is probably better to try both approaches and see what works. Strategy will depend only on available resources since most of the detergents are very expensive. Since a lot more detergent is required during extraction, it may be more economical to extract with an inexpensive detergent and then exchange into others during purification. On the other hand, since doing a test to see best or optimal detergents for extraction will require only lysing small amounts of cell paste, that will cut down on expense of screening during extraction, so that may work out fine as well. As long as you have a nice efficient protocol for screening worked out, you can go for both. A couple of years ago, I was involved in setting up a fast screening protocol for doing this, which we presented in a poster at an NIH PSI Protein Production Workshop. It contains a flowchart which can get you going. It can help to screen a variety of detergents in about 72 hours. Let me know if you want a copy, I'll have to look for it, but can send it to you. Regards, Debanu. -- Debanu Das, SSRL.
________________________________ From: CCP4 bulletin board [mailto:[EMAIL PROTECTED] On Behalf Of Rongjin Guan Sent: Friday, December 21, 2007 11:09 AM To: [email protected] 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
