Hi Crystina,  I see three options. 

1. IP with mouse antibody and probe your Western with a rabbit antibody.

2. IP with your mouse antibody and if you need to use a mouse for Western blot you can use a chain specific anti-mouse secondary antibody. Say your protein of interest is roughly the same size as the heavy chain and would therefore be obscured by recognition of the antibody used for IP, you would use an anti mouse light chain specific HRP antibody, and vice versa.

3. As you mentioned, you could conjugate HRP directly to your primary antibody.

You can also elute your bound proteins by competition if you have a peptide antigen for your antibody, then you shouldn't have any antibody in your SDS-PAGE sample and you can do your Western blot as usual.

I hope this helps,

Nicola

I would suggest rather than crosslinking to HRP (as the crosslinking reaction may effect your enzyme) crosslink your IP antibody to the beads used for IP. We have used this approach before and it actually helps increase the specificity of your IP as well.

Tara Roberts: do you have a protocol for this? Can you share it?

See below for basic method for antibody crosslinking to protein G beads. We did find that the length of time for incubating DMP with the antibody and beads required some optimisation depending on the batch of crosslinker and the antibody.

Crosslinking of Protein G-agarose beads (Millipore, USA) was performed as by Podlaski & Stern (2000), with slight modifications. Briefly, Protein G-agarose beads were washed twice in 1x Sodium Chloride-Tris-EDTA (STE) buffer (10 mM Tris-HCl, pH 8.0, 0.1 M NaCl, 1 mM EDTA), pH 8.5. Antibodies were coupled to Protein G-agarose beads 1 hour at 4°C with periodic resuspension. Pellets were washed thrice with 0.1M borate-NaOH buffer, pH 9. Dimethyl pimelimidate (Sigma, USA) was prepared to a final concentration of 20mM in 0.2M trietholamine cross-linking coupling buffer, pH 8.3 prior to incubation with complexes. After 45 minutes of suspension, 0.2M ethanolamine buffer, pH 9 was added to terminate the cross-linking reaction. Complexes were incubated with 1M glycine, pH 3 to remove unbound antibodies, washed and stored at 4°C with 0.02% sodium azide to prevent microbial growth.