Reprobing of western blot

Darren Tyson 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
> prepared
> 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
1:10000 or
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
worked for
me!)  If you need to use different Abs and you can't use them
simultaneously,
I would recommend using the one that produces a weaker signal first.
Ideally
the two Abs would be from different hosts (e.g. mouse and rabbit) such
that
good secondary Abs would not cross-react.

Cheers,

Darren

--
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)
http://users.primary.net/~darren/





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