Cell lysis, IP, immunoblotting, and antibodies

1. Cells were washed with ice-cold phosphate buffered saline (PBS) 
2. Add NP-40 lysis buffer (with 1% proteinase inhibitor), cell were lysed at 4°C for 30 min
3. Centrifuge at maximum speed,  4°C, 10 min
4. Transfer supernatant to a new tube
5. Mix the samples with the antibody and beads and incubate at 4°C for 3 hours
6. Centrifuge at 2000 ×g, 1 min
7.  Wash the beads with NP-40 buffer 3 times
8. Add the loading buffer and boil at 95°C for 10 min
9. Spin down the beads
10. SDS-PAGE

Solutions:

NP-40 Buffer: 50mM Tris-HCl ,150 mM NaCl, 0.3% NP-40

Antibodies: