HELP needed in western!

Ted M. tedm at darkwing.uoregon.edu
Sat May 27 04:36:23 EST 1995


In article <3piuvr$p0q at newsbf02.news.aol.com>, wzhao80850 at aol.com
(WZhao80850) wrote:

> Hi there,
> I have some problem in western blotting. I am using a high dilution of
> primary antibody for western, i.e. 1:25 (10ug/ml) dilution in TBST with 1%
> milk. Therefore, I have high background in my result. Is there anyway I
> can reduce the background? Also, the protein bands are bended and curved
> on the film. Why is that happened?
> Any suggestion will be great helpful for me, thanks in advance!
> Cheers!
> 
> Wei
> 
> Wei Zhao
> WZhao80850 at aol.com or weiz at protein.bchs.uh.edu
> Tel: (713)669-1091(H), (713)790-4696
> Baylor College of Medicine
> Houston, TX 77030

The other post has some good suggestions, if indeed you are using the
chemilumenescent detection, I have experience with high background being
helped by rinsing between secondary antibody and development with a dilute
solution of NP-40 (say 0.1%). It might not hurt (or help) to rinse between
primary and secondary antibodies, as well. Also proper blocking is
important, proper fit between gel and membrane, perhaps several sheets of
nitrocellulose, and no bubbles. Proper cooling assures bubbles won't form
and possibly degas the transfer buffer, if truly paranoid.
   good luck                                       Ted Michelini



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