Hi Kristen,

I would suggest using an isotype control (IgG or IgM depending on your antibody with the same host as the antibody) and a bead only control to ensure your proteins of interest do not bind the beads. The input should be a positive control showing your protein is in the cell lysate (recommended loading range is 2-20%. I typically load 5% as my proteins are not low-expression). To ensure you are pulling down your protein, you could load 2 gels and probe one for your protein of interest (interactor) and one for the pulldown protein.

Make sure to use different species hosts for the IP and WB, example mouse IP and rabbit WB primary antibodies. Also, using a conformation-specific secondary, heavy chain specific, or light chain specific secondary is helpful to avoid IgG bands that obscure the protein of interest.

Hope this helps,

-Elizabeth