Dear Y B Lin, Measuring detergent has been discussed before on this list - one way is to do thin-layer-chromatography against a set of standards.
http://www.mail-archive.com/ccp4bb@jiscmail.ac.uk/msg12915.html >A strategy for identification and quantification of > detergents frequently used in the purification of membrane proteins > Laura R. Eriks, June A. Mayor, and Ronald S. Kaplan > Analytical Biochemistry 323 (2003) 234–241 To reduce detegent concentration you can reload your protein onto a small Ni-resin or ion exchange column, wash, then elute in a small volume. James -- Dr. James W. Murray David Phillips Research Fellow Division of Molecular Biosciences Imperial College, LONDON Tel: +44 (0)20 759 48895 ________________________________________ From: CCP4 bulletin board [ccp...@jiscmail.ac.uk] On Behalf Of yybbll [yyb...@gmail.com] Sent: Monday, October 04, 2010 3:28 PM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] How to detect the concentration of detergent? Dear all, I want to crystallize a symport transporter, which contains 12 transmembrane alpha-helices. We used Ni-resin column firstly, and then size exclusion. After size exclusion, only one peak, it is very nice. the final condition is 10 mM mes, 100 mM NaCl, 10 mM sucrose, 1 mM DTT, and 0.02% DDM. The CMC of DDM is about 0.008%. However, when we concentrate protein using a concentrator with 50 kDa cutoff, detergent all was concentrated. So final the concentration of detergent should be very high (10 times more than CMC). We don't know how to detect the concentration of detergent. We used these samples to grow crystal. We found almost drops are clear, and the final concentration of protein is about 10 mg/ml. For membrane protein, I think this concentration is high. But for us, we can obtain so high concentration easily. Could anybody tell me how to detect the concentration of detergent? And how to dilute detergent? Thanks all. Y.B. Lin 2010-10-04 ________________________________ yybbll
