Dot blot- not?

Tom Duncan duncant at vax.cs.hscsyr.edu
Mon Jan 27 13:55:45 EST 1997


Warren Gallin wrote:
> 
> In Article <32E95FB5.41C6 at vax.cs.hscsyr.edu>, Tom Duncan
> <duncant at vax.cs.hscsyr.edu> wrote:
> >Of course, you can't apply most denatured proteins in the presence of
> >SDS, since the SDS drastically reduces the binding of most proteins to
> >blotting membranes (nitrocellulose, PVDF).
> 
>     Virtually all Western blotting is done on SDS-denatured protein using an
> SDS-containing buffer, and the results can be both sensitive and
> quantitative, so I wouldn't dismiss simple SDS denaturation and dotting out
> of hand.  It would certainly be worth a try.

For western transfer, the %SDS is usually reduced several fold from the 0.1%
used in Laemmli gel-running buffer; the higher %SDS increases the rate of
transfer of most proteins, but also reduces the binding of many proteins to
transfer membranes (although PVDF seems less affected than nitrocellulose).
Also, even 0.1% SDS is not enough to completely denature some proteins.

>     As far as staying denatured on the membrane, I would guess that the
> proportion of a protein that remains denatured under given blotting
> conditions will be variable, depending on both the nature of the blotting
> conditions and the identity of the protein; once again, I think you have to
> be empirical.

Part of my original suggestion was that the use of nitrocellulose and baking
the blot could be used to covalently fix the protein in the denatured state on
the blot.
	Cheers,
-- 
	Thomas M. Duncan
	Dept. Biochemistry & Molecular Biology
	SUNY Health Science Center
	750 E Adams St,	Syracuse, NY 13210
	Email:	duncant at vax.cs.hscsyr.edu



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