Reprobing of western blot
tysondr.nospam at slu.edu
Tue Jan 27 15:14:36 EST 1998
Frederik Boernke wrote:
> Hi all,
> I tried to detect a GAL4 binding domain fusion protein in a western
> blot using Clontech's monoclonal antibody. Protein extracts were
> according to Printen and Spraque. Protein transfer was verified by
> staining with Ponceau S. Primary Ab was diluted 1:1000, secondary anti
> mouse/HRP-conjugate was diluted 1:100000 (as recommended by the
> manufacturer). Signal was developed using Pierce's SuperSignal ULTRA.
> But....I don't see any bands on the blot even no unspecific ones.
> I thought I'd better repeat that with more "moderate" Ab dilutions.
The primary Ab dilution looks normal... are you sure the 1:100000
dilutionis right for your secondary? That seems awfully low. I'd try
even 1:5000 secondary Ab. Add it directly to your membrane. No need to
strip when there isn't any signal. In fact, I never strip (it has never
me!) If you need to use different Abs and you can't use them
I would recommend using the one that produces a weaker signal first.
the two Abs would be from different hosts (e.g. mouse and rabbit) such
good secondary Abs would not cross-react.
Darren Tyson, Ph.D. Candidate
Cell and Molecular Biology Program
Saint Louis University, St. Louis, MO
tysondr at nospam.slu.edu
(remove nospam. before mailing)
More information about the Methods