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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of the detergent, which has given results identical with the standard procedure using radioactive DDM (T. Warne, unpublished data).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" <[log in to unmask]">[log in to unmask]> 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