Western Blot Double Stain? - (Jul/24/2008 )

I'm wondering if its possible to do the following:
1) stain with my primary antibody (rabbit-anti-protein ~34kD)
2) wash
3) follow with an anti-tubulin primary (mouse-anti-tubulin)
4) Wash
5) Secondary (anti-rabbit)
6) Wash
7) Secondary (anti-mouse)
8) wash
9) detect with ECL (block tubulin from my camera aperature first, then uncover it for a second image)

I'm not sure if this makes sense, but what I'm trying to do is double stain the membrane without quenching. The reason I would cover the tubulin signal first is that it is much higher than my protein of interest. I'd block that, image my protein signal then subsequently image the tubulin signal to normalize. Is this possible?

Shouldn't be a problem. You could combine the 2 secondaries into a single step, if you don't want to keep them separate.

As bob1 suggested, add both the secondaries together, it works.

Hi I was wondering if I could do the same? I have 3 antibodies raised in rabbit which I want to look at seperatley (all between 12-18kDa) and a tubulin control raised in mouse (50kDa). Does this mean I can dual stain each antibody with the tubulin control?

Thanks

This will only work if you know that each of the antibodies do not give non-specific bands (particularly in the regions of your other antibodies). I have done this with antibodies that I know well and are a bit too close in size to cut the membrane. If you are worried about quenching then what you described may not work because with very strong antibodies, I get a 'burn out' centre which is almost impossible to get rid of even after waiting etc. (Need to dilute the ECL then).

What is very commonly done now is to cut the membranes and probe each section with a different antibody - similar to your idea but does not involve antibody mixing. Then each antibody strip can be developed to the best exposure.

Hope this makes sense.