Western Blotting of Membrane Proteins in the presence of SDS

millerbs at LP.MUSC.EDU millerbs at LP.MUSC.EDU
Mon Apr 26 14:45:19 EST 1993


SHAUN,
We usually use 39mM glycine, 48mM Tris, 0.0375% SDS, and 20% MEOH in the
transfer buffer.  We use a Hoeffer semi-dry electroblotting apparatus.
The instructions that came with this instrument suggest 70 minute transfer
at a constant amperage calculated as 0.8 * area to be transferred (cm^2).
This has worked well with several membrane receptors in our lab.  As usual,
proteins of large molecular weight do not transfer as well as smaller ones.
This method has worked well with nitrocellulose or PVDF (Immobilon).
However, PVDF must be wet in MEOH (30 sec) rinsed in water (2-3 min) and 
then equilibrated in the transfer buffer for 15 min prior to transfer.
We transfer gel immediately after running them.

Good luck,

Brad Miller
millerbs at lp.musc.edu



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