Immunoprecipitation Protocol
- Determine the appropriate cell lysate to use for your IP (200-500ul is a good starting point).
- Pre-clear the cell lysate by adding 100ul of either protein A or G agarose bead slurry (50%) per 1ml of cell lysate.
- Incubate at 4°C for 20 minutes on a rocker.
- Remove the protein A or G beads by centrifugation at 14,000rpm for 5 minutes.
- Transfer supernatant to fresh centrifuge tube. Discard pellet.
- Dilute the cell lysate to 1ug/ul total cell protein with PBS.
- Add immunoprecipitating antibody (10-20ug/ml is a good starting point) to pre-cleared cell lysate.
- Gently rock cell lysate/antibody mixture for 2 hours at room temperature, or overnight at 4°C, on a rocker.
- Capture the immunocomplex by adding 60ul of fresh protein A or G agarose bead slurry (50%) and gently rocking for 1 hour, or overnight, at 4°C.
- Collect the agarose beads by pulse centrifugation (5 seconds at 14,000rpm).
- Discard the supernatant.
- Wash the beads with ice-cold PBS three times:
- Add 800ul PBS, rock for 5 minutes at room temperature, pulse centrifuge (5 seconds at 14,000rpm), remove supernatant.
- same as above, but rock for 7 minutes.
- same as above, but rock for 9 minutes.
- Remove as much wash buffer as possible and resuspend the agarose beads in 60ul of 2X reducing sample buffer. Mix gently.
- Boil beads for 5 minutes to dissociate the immunocomplexes from the beads.
- Collect beads by pulse centrifugation (5 seconds at 14,000rpm).
- Transfer supernatant to fresh centrifuge tube. This should be enough to run three lanes on your gel.
Supernatant can be stored frozen at -20°C for later use. Frozen supernatant should be re-boiled for 5 minutes directly before loading on a gel.
*The above information is only intended as a guide. The researcher should determine what protocol best meets their needs. Please follow safe laboratory procedures.