NEPHGE and SDS transfer

Phil Whitehead p_whitehead at icrf.icnet.uk
Wed Mar 27 11:56:25 EST 1996


In article <4celo1$nt9 at cronkite.ocis.temple.edu>,
driska at astro.ocis.temple.edu (Stephen P. Driska PhD) wrote:

> rep at icbr.ifas.ufl.edu wrote:
> 
> : Hey there,
> : 	Happy Holidays and all that.  Posting this question for a friend.  Has
> : anybody done NEPHGE gels and then transferred them under SDS conditions?  I
> : seem to remember some posts about this quite awhile ago, although I think it
> : was just IEF and not NEPHGE.  I would apreciate any replies concerning
> : incubations in transfer buffer or SDS containing buffer before blotting. 
> : Basically, any help would probably be usefull.  
> 
> : Chuck Peterson
> : Whitney Lab.
> : St.Augustine, FL
> 
> 	Do you mean A) transfering a 1-dimensional NEPHGE gel, or 
>                     B) transferring a 2-D, NEPHGE/SDS gel?
> 
> 	I haven't done A, but it should be like doing an IEF except 
> that bands would be in different places.
> 
> 	"B" is not really any different than a 2D IEF/SDS gels.
> 
> 	The only thing that might be different is that with prolonged
> IEF, you might deplete the ampholytes in the gel, whereas in NEPHGE 
> (usually run for a shorter time) you probably haven't lost as much ampho-
> lyte during the run.  It just dawns on me now that you might mean 
> you are worried about ampholytes binding to nitrocellulose/PVDF/nylon, etc.
> In that case, you might have problems.  But let us know what the case is,
> and someone "out there" will probably be able to help.....
> 
> 	good luck, Steve.
> --

I have used this technique ad nausium for cyto keratins with no problems at
all
just use your usual blotting protocol. Ampholines certainly do not stick to
nitrocellulose or PVDF come to think of it so fill your boots get stuck in
and go for it
phil whitehead 



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