Dot blot- not?

Tom Duncan duncant at vax.cs.hscsyr.edu
Fri Jan 24 20:19:49 EST 1997


DSF wrote:
> 
> Is there a way to do protein dot blots with denatured protein
> (that stays denatured on the membrane)? Got a protocol?
> Thanks
> Lynette

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).

You may be able to use heat denaturation before applying the protein to
the blot, as long as your protein does not refold too quickly as it
cools (before or after spotting on blot). Another possibility is
denaturating with urea, and you may be able to dilute the urea several
fold before blotting as long as the protein(s) you're interested in do
not refold too rapidly as the urea is diluted. You would also have to
test how well your protein(s) binds to the blotting membrane in the
presence of urea.

As you noted, a further problem is that, after dot-blotting, some
denatured proteins can refold during incubation of the blot in the
common buffers used for incubation with blocking agents and antibodies.
Although I did not test this for different proteins at the time, one
approach that worked in a specific case involved blotting on a
nitrocellulose membrane and then baking the blot at 80deg (in a vacuum
oven) to denature the protein and covalently link it to the
nitrocellulose to "lock" it in the denatured state. Subsequently, some
MAbs that recognized the protein on western blots also detected it on
the dot blot, but another MAb could not detect the protein on westerns
or on the denatured dot blot, and so probably recognizes a
"conformational" epitope.
The reference:	Adami et al. (1993) Biochem. J. 292, 863-872.
Good luck, and let me know if you are successful.

	Tom
-- 
	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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