To estimate the total concentration of detergent, you can use TLC beside standard amounts of the detergent- see: Analytical Biochemistry 323 (2003) 234�C241 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*
Interestingly this paper also discusses concentration of detergent by ultrafiltration. You need to be clear about total concentration of detergent vs concentration of free detergent. The protein will bind some detergent, up to maybe 1 g/g protein as you approach CMC, so if protein is 10 g/l you might have 10 g/l total detergent (100 x cmc) and the concentration of the monomer still be below CMC. I take it your 0.008% is concentration in the column buffer, so assuming the protein is equilibrated with that, it is the free detergent concentration. If the pass-through from ultrafiltration contains the same concentration, it means the detergent is not really being concentrated, only the protein-bound detergent is being retained by the filter. Even if all the free detergent is retained, it might be an insignificant increase in the total concentration, and the free concentration could be "buffered" to below CMC by protein binding. eab yybbll 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
