Protein sequencing from a western blot

Mick_Partis_at_HRIW05 at HRI.BBSRC.AC.UK Mick_Partis_at_HRIW05 at HRI.BBSRC.AC.UK
Thu Nov 9 07:25:22 EST 1995


     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.
     
     >2. can it be done from an SDS-PAGE gel as well as a native PAGE blot?
     
     Yes.  I usually run it this way - almost any PAGE system is suitable
     
     > Any references would be appreciated!
     
     P. Matsudaira (1987) Sequence from picomole quantities of proteins 
     electroblotted onto polyvinylidene difluoride membranes J. Biol. Chem 
     262:10035-10038
     
     Millipore have a number of leaflets and application notes on their 
     Immobilon PVDF membranes which are useful
     
     Applied Biosystems have stuff on their ProBlott PVDF membranes.
     
     Mick
     
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     Standard disclaimers etc.......




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