Hi Katarzyna, Yes, membrane proteins can be purified in DDM at ~1-2xCMC. Since its CMC value is very low, at the extraction step you need to use a much higher concentration up to ~100x CMC. During different rounds of purification, you can bring the CMC level down to 1-2x CMC and even try detergent exchange in the last step, although it can be difficult to totally exchange out the DDM due to its low CMC. Also use a 100 kDa MWCO concentrator since the DDM micelle size is large.
In general, DDM is a reasonable choice for extraction, purification and crystallization setups, but it will be worthwhile to screen a panel of different detergents for extraction and purification for your particular target. In 2005, we set up a fast protocol for screening a panel of 18 detergents in 48-72 hours, http://smb.slac.stanford.edu/~debanu/posters/012605_PPCW2005_DDAS.pdf, which includes suggestions for starting concentrations for extraction. There are also some papers on detergent screening, for example: http://www.ncbi.nlm.nih.gov/pubmed/18988031 http://www.nature.com/nprot/journal/v4/n5/full/nprot.2009.27.html (Stroud lab) Thanks, Debanu. -----Original Message----- From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Katarzyna Rudzka Sent: Monday, March 26, 2012 10:17 AM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] DDM Hi All, Has anyone had any luck purifying membrane proteins with DDM (n-dodecyl-D-maltoside) using concentration of detergent ~1-2 x CMC ? (Its CMC is very low: 0.009%). I would like to keep it as low as possible, so I don't have too much DDM around when I get to the crystallization step. I wonder If the amount of detergent sufficient for the protein extraction has to be determined experimentally for each protein or maybe there are some good rules of thumb. I appreciate your help. Thanks. Kasia Katarzyna Rudzka, Postdoctoral Fellow Department of Biophysics and Biophysical Chemistry Johns Hopkins University, School of Medicine Baltimore, Maryland 21205 USA
