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