Andre Hamel hamel at ccu.umanitoba.ca
Fri Feb 19 19:11:43 EST 1993

In article <9302171757.AA16598 at net.bio.net> ORSULIC at UNCVX1.BITNET writes:
>I need information regarding transfer of proteins to PVDF membrane for seq.
>What buffer conditions, volt/current and how to stain ponceau or coomassie.

We've used regular 20% methanol in SDS PAGE (Laemmli) buffer at 4oC for
electroblotting onto PVDF, at about 100 milliAmps for about an hour for
proteins less than about 20 kDa in 15%T acrylamide gel. For larger
proteins use longer time. Pre-wet the PVDF membrane in 100% methanol
before use! Use flat (Millipore filter type) forceps, gloves can leave
residue on membrane.

Remember to NOT have UREA in the SDS PAGE gel nor in any other buffers
coming in contact with the protein, as this will cause poor sequencing.
Stain with FRESHLY made 0.1% coomassie in just 50% methanol for about 15
 min to 1/2 hour, then wash few times for few minutes each.

You can send the cut out membrane band piece stained as is, Coomassie does
not interfere with protein sequencing.


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