protein band excision/concentration

Peter pxpst2 at SPAM.SUXS.unixs.cis.pitt.edu
Sat Aug 1 12:03:11 EST 1998


In article <6ptm39$vsa$1 at nntp4.u.washington.edu>,
pearton at saul5.u.washington.edu (D. Pearton) wrote:

 
> As an alternative, if you're trying to identify the peptide why not
> silver stain followed by in-gel digestion and elctrospray MS/MS?

Sounds good but if the protein is any larger than a 1-2 kd then you will
have a mess with analysis.  Do able but NOT easy.  Also Siver with MS is
not a great combo.
> 
> Yet another method might be to blot onto PVDF, coomaasie stain and
> N-terminally sequence the band.

Actually this is the most reasonable and if you have access to the above
MS MS then you should be able to obtain the total mass of the protein, not
just the N termeinal.
> 
> There are also charged versions of PVDF that are designed for elution of
> proteins.  I must admit I've not had that much success with them.
> 
Actually Ithink that you simply dissolve the membrane away and precipitat3
then redisolve. The reference is not handy but I will post it later(on
monday).
Also I have recently come accros a really cool procedure that "prestains
the sample and aloows you to see you bands so you eliminate the loss to
diffusion that would accompanty yhe staining step with either silver or
commassie.  An the smaller your protein the more you lose to diffusion
even with fixing.  The guy that did the work was a German named Schlaegger
and he also published the procedure for continuous Tris Tricine PAGE that
is one very cool technique for the resolution of small proteins.  If you
are working with proteins less than 100 kd there is no substitute for this
protocol.  It is in my opinion giving binoculars to the Lamelli method.

Peter

-- 
"Don't you eat that yellow snow
            watch out where the Huskies go"    FZ

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