Western blot

Martin Blankfard Mblank at kpl.com
Thu Jun 22 16:17:04 EST 1995


In article <3s7nlj$138v at hearst.cac.psu.edu>,
   Aida Cancel <axc19 at psu.edu> wrote:
>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
>


Your problem here may not be just your blocking solution but also you 
conjugate or conjugate concentration.  In most cases I block my membranes with 
2% non fat dried milk.  But in your case this could be a real problem.  I 
would block your membrane in 2 % fish gelatin or 2% egg albumin.  In addition 
you may want to be sure you are not using your conjugate to concentrated.  
Determine the best conjugate dilution to give the greatest signal with the 
least background.  In most cases this resolves the problem of background.  The 
other place to look is your wash solution.  Sometime you need to add some mild 
chaotropic agents to your wash solutions to remove nonspecific antibody 
binding.  For this I would increase the salt concentration (NaCL) of your 
PBS-Tween to maybe .5M.  I usually perform 3 , 3 minute washes after primary 
and conjugate incubations.  In addition always rinse the strips in DI water 
before adding the substrate.  Here again rather than DAB you might want to 
think about using TMB, its a better substrate for membranes in my opinion.

If you continue to have problems I can supply most of the reagents discussed 
above including the conjugates, wash and substrate for you to try.



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