I have been performing CoIP experiments with the ThermoFisher Pierce classic Magnetic IP kit. I havent couple my antibody to the beads, instead incubated lysate, antibody and beads in a tube overnight at 4oC on a rotator.
The next day I have collected supernatant samples and tried various different wash steps/combinations with buffers of 150 mM NaCl and buffers of 500 mM NaCl. However with all combinations of washes, I am getting unspecific bands at 150 and 200 kDa as well as the bands at 50 and 25 kDa for the heavy and light chains of anitbodies.
Can anyone suggest what these bands could be? Or what optimisation steps may be successful at minimising them?