Hi everyone!
I would like someone to help me with some Western blots that I am trying to work
out. I can successfully transfer proteins extracted from the reproductive tract
(testis, epididymis, prostate, seminal vesicle, bulbourethral gland etc.) to
nitrocellulose membranes. This I am sure, because I detect the proteins with
Ponceau S staining. My problem now is that I am having troubles with a detection
procedure. I started using PBS-0.1% Tween 20 + 5% Normal goat serum to block
(overnight at 4 C)...all subsequent washes and incubations were done with PBS-Tween
+ 1% NGS. Finally using DAB to develop the reaction. The result: huge, MASSIVE
background.
I then tried 5%BSA (PBS_Tween) as a blocking agent and in all my washes..the problem
was that this time I knocked out all signal. Sooo, I tried ECL, using 0.1% milk,
PBS-T20 followed with a second block with 0.5% BSA in TBS T20. Again I can not
detect any signal.
Since then I realized that probably I can not use milk because the protein that I am
studying is found in milk. Thus, the antibody recognizes the protein in the
blocking step.
Anyone has any suggestions? I have called a couple of places that deal with
extracted proteins fromt the reproductive tract and they all seem to agree that
there is always too much background in their Western blots. I am getting desperate.
Any help will be appreciated,
Aida
axc19 at psu.edu
