conjugated antibodies in immunoprecipitation - (Jan/04/2013 )

Ok, I have a couple of questions about using conjugated antibodies in both Western Blotting and immunoprecipitation.

We study a cell surface protein, and use a primary antibody followed by a FITC conjugated secondary antibody to cross-link the protein on the cell surface, causing it to form clusters that we can then see using immunofluorescent microscope. In order to provide consistency, I want to use the same two antibodies on the cells we are using for western blots, to investigate the proteins and pathways activated upon the surface protein cross-linking.

I have used this on western blots before, and not noticed any additional staining on the ECL film that I was worried may occur with the FITC fluorophore, so Iam not worried about it affecting that anymore.

What I am more worried about is the immunoprecipitation experiments.


1-Firstly, will cross-linking of the surface protein with two antibodies (A-primary mouse anti-human, and B-secondary goat anti-mouse) interfere with the use of a third antibody (C-mouse anti-human, different antibody to A) in the immunoprecipitation? Or will the whole complex bind to the Protein-G beads anyway due to the previous two antibodies used?

2-Secondly, The protocol states to use an isotype control antibody to 'clear' the lysates of stuff that may bind non-specifically to the protein-G bead/antibody complex. The only control antibodies we have in the fridge are conjugated ones, to FITC, PE, etc, for use in FACS controls. Would it be ok to use a conjugated control antibody in these experiments, or would the conjugation get in the way of the epitopes required to bind protein G?

Thanks, and thanks to any people around this early in the year to answer!

1-Protein G will bind your secondary antibody and your primary antibody used to induce clustering of your cell surface protein.
2-Fluorophore-conjugated antibodies would be expensive isotype controls. It seems like the answer to question #1 doesn't leave room in your prototocol for preclearing, however.

In my experience pre-clearing is done using the beads only in the lysate, no antibody. You can add some BSA or something similar as a blocker, which would equate to using an isotype antibody. For polyclonals it is common to use pre-immune serum (e.g. normal rabbit serum) as the blocker in these sorts of experiments.

I was just asking about the conjugated antibody controls as we already have those and they are not being used, so it would save spending another £100+ on more non-conjugated controls.

I will take your advice bob1 and try the clearing with just the beads then, and hope that is enough.

Thanks!