Protein sequencing from a western blot

Warren Gallin wgallin at gpu.srv.ualberta.ca
Thu Nov 9 11:46:24 EST 1995


In Article <47srvi$obk at mserv1.dl.ac.uk>,
<Mick_Partis_at_HRIW05 at HRI.BBSRC.AC.UK> wrote:
>     Lynne Whitehead wrote....
>     
>     >I am interested in learning more about protein sequencing from western 
>     >blots.
>     
>     >1.  is this method reliable?  How much protein is required .....
>     
>     Depends on the systems involved.  If you have a clean extract with no 
>     adjacent protein bands to cross-contaminate your protein of interest, 
>     and you use good transfer media (PVDF membranes, transfer buffer 
>     without glycine), then I have found it to be O.K.  As to sensitivity, 
>     it depends on the sequencer:  a gas-phase sequencer will give about 10 
>     residues of sequence if the band was stainable by coomassie blue in 
>     the gel.  The usual quote for ABI gas phase sequencers is 10-100pmole: 
>     this is microgram range for most proteins. Obviously, the more protein 
>     one has on the blot, the more sequence one gets back, and the 
>     reliability increases.

Just a minor point.  You say Western blots.  You certainly can not sequence
the protein if the membrane has actually been blocked and visualized with
antibody (Western blotted); the blocking protein and antibody would be the
major protein on the membrane.  Sorry to be so pedantic.

Warren Gallin,
Department of Biological Sciences, University of Alberta
wgallin at gpu.srv.ualberta.ca



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