Hi YB, There is a nice new paper online, dealing with this subject:
http://www.ncbi.nlm.nih.gov/pubmed/20850411 Cheers Phil Am 04.10.10 17:09 schrieb "Owen Pornillos" unter <o...@scripps.edu>: >>> Could anybody tell me how to detect the concentration of detergent? > From Butler et al. (2004) J Mol Biol 340: 797-808 > > The concentration of DDM was determined by a colorimetric assay that detects > the sugar component of the detergent, which has given results identical with > the standard procedure using radioactive DDM (T. Warne, unpublished data). A > 60 μl sample containing 4–16 μg of detergent was mixed briefly with 300 μl of > 5% (w/v) phenol and then 720 μl of concentrated sulphuric acid was added > followed immediately by vortex mixing for five seconds. The samples were left > to cool for 30 minutes and then the absorbance at 490 nm was measured. A > typical standard curve contained six samples containing between 0 μg and 20 μg > of DDM, which gave a straight line (r2>0.97). > > HTH, > > Owen > > > On Oct 4, 2010, at 10:42 AM, Van Den Berg, Bert wrote: > >> Hi YB, >> >> For membrane protein crystallization it is common practice (although not >> always necessary) to dialyze the protein after the final concentration step >> (against GF buffer). The problem with DDM is that dialysis is slow due to the >> low cmc, and in general it is advisable to finish the prep and set up drops >> as quickly as possible. I would not dialyze more than 1 x O/N, although if >> your protein is really stable you could try longer. You could also try 100 >> kDa cutoff concentrators, as these may allow passage of empty DDM micelles. >> As for measuring the detergent concentration, in the case of DDM and other >> sugar-containing detergents you could do a sugar (Fehling’s) kind of assay. >> I’m not sure if anything has been published, but it should be fairly easy to >> do. You could also try TLC, but this may be less accurate. >> >> Also, 10 mg/ml is not high at all (although its a good starting point), and >> you should try much higher if most drops are clear: try 50 mg/ml and see what >> happens. >> >> Good luck, Bert >> >> >> On 10/4/10 10:28 AM, "yybbll" <yyb...@gmail.com> wrote: >> >> >>> 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 >>> >>> >> >> > >
