>Date: Wed, 14 Mar 2001 07:49:13 -0800 (PST)
>From: sel dom <msu48843@yahoo.com>
>Subject: Re: membran protein
>To: Bob Keefe <keefe@wadsworth.org>
>
>Bob
>thank you for your reply, I am trying to remove the
>urea and ad 0.1% b-octyl, but this did not help as I
>dialyse from 4M urea to 3M then to 1.5M but when I
>move to 1.5M I can see the protein precipitate in the
>dialysis tube, also I add to my urea free buffer (last
>dialysis step) some ligand, I use 1M or 3M urea in the
>lysis buffer as i express in bacteria nad I use
>affenity column with ligand attached to teh reisin
>then I elute with higher ligand concentration, I
>always see chaparon protein on the gel with my protein
>and I am trying to get hold of clean protein for
>crystalization.
>--- Bob Keefe <keefe@wadsworth.org> wrote:
>> 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
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