Double labelling in IF - (Sep/07/2009 )

Hello,

I made a mistake in selecting the two scondary antibodies for my immunofluorosence study. One is a goat anti-mouse while the other is a donkey anti-goat (can you see the problem yet?). I got the goat anti-mouse one as a present from another lab and it wasn't well labelled. I've since discovered the error and looking back over my images, there doesn't appear to be co-localisation of the signal which is a hopeful sign that they're not cross-reacting.

My qustion therefore is why? Is it possible the the fluorophore on the goat anti-mouse is blockinng the binding site of the donkey anti-goat? Has anyone made a similar mistake and got away with it? General advice is also appreciated.

Mr Jefferson on Sep 7 2009, 04:20 PM said:

Yes, it's certainly possible. You can always try to go on the vendor's site and see what sequence their antibody targets. It's sometimes proprietary, but they often say it on the website with the antibody info.

Have I gotten away with it? Nope. I think that's one of those mistakes that a lot of people will make once then never do it again.

Oh, and check if your DaG is monoclonal or polyclonal. If it's polyclonal then there's almost no way you just got lucky, since it would target multiple sites on the GaM.

Cheers,
-Carlton