PVDF contra gel extraction for protein sequencing ?

Peter pxpst2 at unixs.cis.pitt.edu
Tue Nov 3 10:16:20 EST 1998


In article <71mnkl$9ro$1 at fu-berlin.de>, "Oliver Politz"
<politz at medizin.fu-berlin.de> wrote:

> I would like to get some opinions about advantages / disadvantages to =
> use either gel extraction of proteins or blotting them on PVDF membrane =
> to perform tryptic digests for further HPLC separation and =
> microsequencing. My problem is that I have to get the protein of =
> interest out of a number of faint coomassie bands. I am thinking of =
> using the electro-eluter  module for the Biorad Miniprotean cell (any =
> experience out there).

PVDF can be great but there is one problem.  If you perform tryptic/V8
digests, some of the hydrophobic fragments my not come of your membrane
even under basic conditions.  Also when staining with CB, you may
dehydrate some of your amino acids making analysis more ambiguous via MS
analysis.  Also if you band only stains slighly with CB, then you may be
below the detection limits of a HPLC sequencing apparatus, but you should
be fine using MS MS machines.  Often it is prefered to stain with Ponceau
Red.  But P-red is a horribly insensitive stain and sucks rocks with
heavily glycosylated proteins.

Hope this rambling helps.
Peter

-- 
"don't you eat that yellow snow
       Watch out where the huskies go"  
                             FZ



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