Coomassie staining before electroblotting?

Chris Kafer ckafer at iastate.edu
Thu Jun 12 12:16:23 EST 1997


Thanks for the info!  This was always my belief.  Interesting that the
coomassie will transfer but not the protein.  My labmates (and PI)
point to the nitrocellulose with blue bands as proof that it works.  I
will be interested to see if they actually detect any protein.

Since we use the older tank style blotter with lots of buffer and long
transfer times, vs a semi-dry apparatus, I wonder if this helps
re-solubilize the proteins.

I will pass along the collective wisdom of the replies I receive,
but...


On Thu, 12 Jun 1997 16:10:26 +0200, fof1 at chclu.chemie.uni-konstanz.de
(Frank O. Fackelmayer) wrote:

>In article <339f82f0.226556441 at news.iastate.edu>, ckafer at iastate.edu
>(Chris Kafer) wrote:
>
>> Can/should an SDS protein gel be stained with coomassie before
>> electroblotting onto nitrocellulose which will be followed by
>> detection with antibodies and protein A?  I thought not, but am not
>> sure why!
>> 
>> Thanks!
>> 
>
>Hi Chris,
>Don´t stain your gel with coomassie blue before electrotransfer. The
>reason is that the acetic acid/methanol mixture fixes (precipitates) your
>protein in the gel, and you cannot solubilize it again efficiently.
>Nevertheless, I tried it several times, with the interesting result that I
>got transfer of the stain to the membrane, but not of the protein....
>
>If you definitely NEED to stain your gel before transfer, use a
>non-fixating stain, like copper chloride or the commercially available
>"RotiWhite" from Carl Roth GmbH, Karlsruhe, Germany (no affiliations).
>
>Hope this helps,
>Frank

************************************************************************
Chris Kafer	www.public.iastate.edu/~ippm	       PGP Key available
		www.public.iastate.edu/~thorn
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