Immunoprecipitation
Take cell dishes out of incubator and pre-cool on ice.
Rinse cells gently with ice-cold PBS for twice.
Scrape cells and spin down @ 500 rpm, 10min, 4 °C.
Add 500l ul IP lysis buffer to each cell pellets (upto 2E+7 cells).
Rotate for 2h at 4°C.
Centrifuge 14,000rpm for 30 min.
Save supernatant.
(Optional) Roughly determine protein concentration with OD280.
Adjust all samples to the same concentration.
Determine amount of beads needed (Rockland True Blot IP beads; 20 ul beads per 500ul sample).
Wash beads in 1ml lysis buffer for twice.
Spin down @500g, 30s.
Resuspend pelleted beads in 1mg/ml BSA , rotate @ RT, 10min.
Remove BSA and wash beads in lysis buffer for 3 times.
Resuspend pelleted beads 1:1 with lysis buffer.
Save 40ul of IP lysate per sample as input.
Add primary Ab and beads to IP lysate.
Rotate @4 degree for overnight.
Pellet beads @ 500g for 30sec @RT and discard the supernatant.
Wash beads in 1ml ice-cold lysis buffer.
Repeat 3 times.
Resuspend beads in 30ul of 2× loading buffer.
Boil the beads for 5min.
Centrifuge @ RT for 1min.
Save supernatant and perform SDS-PAGE.
