Dot blot- not?

[Tom Duncan]

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: dun...@vax.cs.hscsyr.edu

[DSF]

[Warren Gallin]

In Article <32E95F...@vax.cs.hscsyr.edu>, Tom Duncan
<dun...@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.
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.
Warren Gallin
Department of Biological Sciences
University of Alberta
Edmonton, Alberta T6G 2E9
Canada
wga...@gpu.srv.ualberta.ca

[Tom Duncan]

Warren Gallin wrote:
>
> In Article <32E95F...@vax.cs.hscsyr.edu>, Tom Duncan
> <dun...@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,