Hi all,
Do any one of you are mixing 2 primary Ab's for western blotting protein detection.
I got two different MW proteins , got primary Ab's against them (both anti Mouse).
I am thinking of mixing them together to save some time.
What do you think?

I have never heard of mixing antibodies for Westerns, though I have seen 7 or 8 mixed together for staining cells. It might work, but it seems risky and since antibodies are expensive may waste antibody and may waste time if it does not work.

Anyway, if your proteins are of very different molecular weight, I would suggest Ponceau staining your nitrocellulose membrane, mark the bands in the ladder lane with a pencil (some permanent inks work, too), then cut the membrane into two pieces with a clean razor blade (cut it on a very hard surface such as a pane of glass). The membrane pieces can be blocked together, primary antibody in separate containers, washed separately, then secondary antibody in the same container (since they are both mouse antibodies).

Please check your primary antibodies to see if their isotypes are different. If they are different, you can use our anti-mouse isotype -FITC ( green), Cy3 ( red ) or
APC (blue) to do the multicolor western blotting. (Picture available, but too large for this site.)

Best regards,

Stacey Gwin
VP New Business Development
SouthernBiotech
U.S. & Canada Toll Free: 800.722.2255
Office Phone: 205.945.1774
Direct Phone: 205.601.8544
Fax: 205.945.8768
Email: sgwin@southernbiotech.com
Website: www.SouthernBiotech.com

Thank you for you input Stacy
I will try it.
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Guy