2 primary antibodies, both binding actin...they shouldn't be :( - (Jul/02/2012 )

Hey all,
I did a western the other week and used two primary antibodies (mouse anti-actin and rabbit anti-IL7R) and then used two secondaries, (goat anti-mouse, and goat anti-rabbit). Both secondaries are labelled with IR dyes, the anti mouse is 680nm, the anti-rabbit is 800nm.

I admit, I transferred the proteins to PVDF, then incubated with both primaries together, and then both secondaries together. I ended up with a lovely yellow band at the size of actin, and nothing at the size of IL-7R. Have I screwed it up? Should I incubate everything separately? Which order would be best? I figured since I had different species and targets I would be ok to incubate it all together....

have you confirmed that the antibodies worked individually and at the concentrations you used? if so, then it should have worked as you envisioned.

if not, then you may have to tweak the protocol.

We know that both secondaries, and the anti-actin work fine, but I'm not sure about the anti-IL7R...in all honesty I have no idea how long it has been in the lab. That was part of the reason I did the western. I just wanted to make sure that I hadn't inadvertantly screwed things up with my simultaneous incubations.....

If you aren't sure of the IL7R antibody, you should verify that it works as it's supposed to before you do simultaneous incubations. Also, if the antibody has been around a while (and especially if you don't know that it has always been stored properly, etc.), it may just have gone off.

Also, do you know for sure that the sample you used expresses IL7R?