Immunoprecipitation (IP)

Lyse cell pellets (5-7mg) in 500µL IP lysis buffer containing IP base buffer supplemented with 1x cOmplete, EDTA-free protease inhibitor cocktail and 0.1µL of benzonase and incubate samples On ice for 0h 30m 0s. Mix the sample by inverting the eppies gently every 5 min.

Wash anti-HA beads with 500µL of bead equilibration buffer.

Repeat step 2 twice.

Centrifuge the cell lysates at max speed for 0h 10m 0s at 4°C.

Carefully transfer cleared lysates into 2 ml eppies and take 50µL from each tube for “Input” samples.

Gently add 1000µL of IP base buffer containing 1x cOmplete, EDTA-free protease inhibitor cocktail to the rest of each sample to dilute out the detergent.

Incubated the diluted cleared lysates with the anti-HA magenetic beads on a rotary mixer for 3h 0m 0s at 4°C.

Collect beads on a magnetic rack and aspirate the unbounds.

Wash with 1mL IP wash buffer.

Repeat steps 7-8 another 4 times.

Elute with 25µL elution buffer by boiling at shaking at 99°C for 0h 10m 0s.