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 .
