Dot blot- not?

Warren Gallin wgallin at gpu.srv.ualberta.ca
Fri Jan 24 18:46:33 EST 1997


In Article <32E95FB5.41C6 at vax.cs.hscsyr.edu>, Tom Duncan
<duncant at vax.cs.hscsyr.edu> wrote:
>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).

    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
wgallin at gpu.srv.ualberta.ca



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