Re: EBlot

Katheryn Resing (Katheryn.Resing@Colorado.EDU)
Wed, 15 Apr 1998 10:31:45 +0000

I have used two approaches in enhancing transfer of difficult proteins.
Putting 1-5 mM thioglycolate in the SDS-PAGE running buffer often causes a
big improvement in transfer efficiency (presumably it quenches peroxides in
the gel that cross-link unusually reactive proteins with the acrylamide).
In another case, I only wanted immunoreactivity, so I put a small amount of
protease in the filter paper on the back side of the blot. This
significantly enhanced transfer of larger proteins (in this case about
750,000-1,000,000).

Katheryn Resing

>A client has a 100kDa protein that does not electroblot from 7.5%
>acrylamide gels using CAPS/MeOH or Towbin/MeOH buffers. This is a selective
>phenomenon as all other proteins in his sample as well as ~100kDa standards
>are successfully blotted. We have reduced methanol concentration and
>included low concentrations of SDS without success. Longer blotting times
>are also of little avail.
>
>Any suggestions out there?
>
>Thanks,
>
>Larry Dangott
>
>
>
>
>
>Larry Dangott, Ph.D.
>Protein Chemistry Laboratory
>Texas A&M University
>College Station, TX 77843-3255
>(409) 845-2965