Olga,
What do you mean by "0.15 b-octyl glycoside"? i.e., what are the units to
this concentration?
The CMC of octyl glucoside is approx. 0.73% (w/v), or ~25 mM (in 50 mM
Na+). If this detergent is your detergent of choice, you may have to
increase the concentration (above the CMC) to see a stabilizing effect on
your receptor. This could become a costly endeavor as octyl glucoside is
not cheap, and has a relatively high CMC compared to, say, the
polyoxyethylenes (like Triton X-100). Your protein wants to be in a
hydrophobic environment, and once you remove the urea, you'll need to
provide some type of substitute for the phospholipid environment it was
extracted from. What you use, of course, will depend upon what you want to
do with your extracted receptor after you've removed it from membranes. If
you haven't done it already, try some other (non-ionic) detergents to
evaluate their effect on minimizing/eliminating protein precipitation.
I've had good luck in the past adding glycerol (5-10% (v/v)) to
detergent-solubilized membrane proteins to stabilize enzymatic activity as
well as minimize precipitation while carrying out subsequent column
chromatography. Careful though - too much glycerol will make solutions
quite viscous - making the concentration of your sample using one of those
pressurized stirred-cells problematic. It (glycerol) is inexpensive, and
easy to remove by dialysis if necessary.
Hope some of this was useful,
Bob
At 08:05 AM 3/13/2001 -0800, sel dom wrote:
>I wonder if any one can help, I have a membrane
>protein in 5M urea but to remove the urea it
>precipitate, it is a ligand binding domain protein
>with the ligand bind to it, in 2M urea is OK but less
>than that it come out of solution even I am using 0.15
>b-octyl glycoside as none ionic detergent but it did
>not help
>
>Olga Matthew
>Yale University
Robert G. Keefe, Ph.D.
Wadsworth Center/NYS Dept. of Health
Genomics Core Facility
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