protein purification

Peter pxpst2 at vms.cis.pitt.delet.edu
Mon Dec 15 12:02:02 EST 1997


In article <6729qf$eg5$1 at enyo.uwa.edu.au>, karenmk at cyllene.uwa.edu.au
(Karen Kroeger) wrote:
> I am trying to sequence peptide fragments generated from tryptic digests.
> I want to purify a the protein by cutting it out of an SDS gel then
> digesting this cut out protein with trypsin, then run this digested
> product on a second SDS gel. Does any one have a detailed method on how to
> do this procedure, any help would be appreciated.  Eg.  can you do in gel
> digestions, what special conditions are required etc.  Any information
> please.  Please reply by email.

You will have big problems with this strategy.  By the time you finish
with all your steps you will have lost  or destroyed your protein.
First you should run the gel and cut out your band 
2)trypsinize your sample in the gel.
3)sample can now be applied to a MS sequencer and the fragments can be
IDentified.


Go to the Lybrary and get
Annalytical Biochemistry @@$, 75-82 (1995)
Internal sequences from proteins in Polyacrulamide gels



BTW, this is NOT trivial and you are in for the pain of your life

Peter Pediaditakis



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