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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 
     >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 
     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.partis at bbsrc.ac.uk        Horticulture Research International
     Standard disclaimers etc.......

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