Hi Noel,

I would highly recommend that you cross-link your proteins, otherwise, with such a concentration of detergent (necessary to get the proteins out of the membrane), you will for sure destroy any non-covalent interaction.

I used to use DSP (ThermoScientific) for protein crosslinking. i also used BS3 to crosslink my Ab to my Prot G beads...

Another thing that i found working better is magnetic beads instead of sepharose or agarose beads... this way you don't have to do the spinning steps, then less harsh for the weakly bound proteins...

Hope that helps...

I agree with Vanessa, you can also used (BSOCOES, Pierce), a membrane-permeable cleavable crosslinker.

I think you may try to incubate at 4 ℃ for overnight instead of 4 hours.  

Crosslinking as mentioned by Vanessa might help.

I would lyse a 10cm dish with no more that 300ul of lysis buffer to keep samples concentrated. that should help with band intensity and reduce how much antibodies are required per volume of lysate. Secondly if you are concerned about protein degradation, add as proteinase inhibitors and carry out entire experiment on ice and at 4oC. e.g. scrape cells with ice-cold lysis buffer on ice keep tubes on a rotator in the 4oC cold room to lyses. carry out all washes on the 4oC rotator and in a cold centrifuge.