Immunoprecipitation protocol
Chuyu Chen
Abstract
Immunoprecipitation procedure for brain tissue material
Steps
Add 750ug tissue in 1ml IP buffer and antibody, incubate 4°C 2hrs on tube rotator
Prepare ProteinA/G beads 30ul/sample. Pool all beads in one tube to perform wash
Centrifuge 4°C 13000g 30sec, remove supernatant
Resuspend beads with 80ul PBS/sample. Centrifuge again at 13000g for 30sec.
Repeat wash 3 times.
Remove supernatant with fine tip
Resuspend beads with 30ul PBS/sample
Add 30ul beads to protein sample
Incubate 4°C overnight on tube rotator
Centrifuge 4°C 4000rpm 4min, remove supernatant
Wash with IP buffer (+ protease inhibitor) 800ul/sample
Repeat 3 times, total 4 wash
Remove supernatant with fine tip
Add 1x loading buffer 25ul and 5ul reducing agent. 75°C 5min
Centrifuge 4°C 4000rpm 4min, collect supernatant for western blotting
