Kasia, A lot of people uses DDM to purify membrane proteins, not a lot of people crystallises them. If you want to crystallise a protein purified in DDM, then you should use LCP. If you go for vapour diffusion, you should exchange the DDM for a detergent with a smaller micelle size otherwise you might get crystals but it is difficult to get good diffraction. Try mixed micelles for example. Typically use 0.05% DDM during purification and use 100kDa cut-off membranes in order to prevent detergent concentration. For extraction it depends on your protein and expression system but you can see in the literature values between 0.5-2% being used successfully. Good luck. Cheers, Joao
Joao Dias, Ph.D. Senior Scientist Heptares Therapeutics Ltd BioPark, Broadwater Road, Welwyn Garden City, Herts, AL7 3AX UK This document contains company confidential and/or proprietary information. It is intended for the exclusive attention of the addressee(s) above and should not be copied or disclosed to any other. If you have received this transmission in error, please make no use of its contents and contact the sender. [cid:image6c7082.GIF@43484746.4ab9695e]<http://www.heptares.com> The information in this e-mail is confidential and may be legally privileged. It is intended for the exclusive attention of the addressee stated above and should not be copied or disclosed to any other. If you have received this transmission in error, please make no use of its contents and contact the sender. Contact and registered office address: Heptares Therapeutics Limited, BioPark, Broadwater Road, Welwyn Garden City, Hertfordshire, AL7 3AX. From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Katarzyna Rudzka Sent: 26 March 2012 18:17 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
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