Phil Calvert wrote:
>> In article <5rnko6$qm8 at falcon.le.ac.uk>, "Dr E. Buxbaum" <EB15 at le.ac.uk> wrote:
>> > The phenol can be removed by extraction with diethyl ether (2 times twice
> > the volume of your sample). This will leave proteins in the small amount
> > of water which was dissolved in the phenol.
>> Thanks for the reply. Yes, I am aware of that method. I even used it once
> about a year ago. However, I don't really trust it. When I used the
> method I got a lot of fluffy precipitate which wasn't expected (the paper
> first describing the method doesn't mention it). Although I don't have
> proof, I suspect that the diethyl ether may be denaturing the protein.
> Maybe not all the protein, but it is plausible that some of the protein
> could be denatured by this procedure.
>> --
> ------------------------------------
> !-----------Phil Calvert-----------!
> !---calvert at eos.ncsu.eduX-NoSpam---!
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If you're getting a fraction that contains protein and can get rid of
the organic solvents, you can use a protease to ask whether or not the
material is protein-- a good enzyme for this purpose is proteinase K,
which is stable to SDS and will digest everything down to dipeptides or
amino acids. For this approach, you might do even better by first your
precipitating your cell extract with ice-cold trichloroacetic acid (5%
final), washing with ethanol and/or ether to remove the TCA, then
dissolving in buffer for the proteinase (e.g., 20 mM Tris-HCl, pH 7.6,
0.5% SDS). Use proteinase K at 50 to 150 ug/ml and incubate a few hours
at anywhere from 37 to 55oC. Compare acid-precipitable cpm before and
after proteinase treatment (but beware, SDS interferes with TCA
precipitation, so dilute samples before precipitating)
I hope this helps.
