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unpublished data).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
