Western blot

Aida Cancel axc19 at psu.edu
Tue Jun 20 19:04:03 EST 1995


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




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