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