Immunoprecipitation (IP)

1. Culture cells (MEFs etc) in plate with 10 cm in diameter, 37C, 5% CO2 incubator.
2. Starve the cells overnight at least 18 hours with medium containing no serum.
3. Treat the cells with insulin (100 nm) or others for a time period of 30 min.
4. Wash the cells with cold PBS twice.
5. Add the cell lysis buffer 900 ul.
6. Place the cells at 4C cold room for shaking for 30 min.
7. Harvest the cells into 1.5 ml eppendof tubes.
8. Centrifuge the tubes at 14,000g for 10 min at 4 C.
9. Transfer the supernatant to the fresh tubes and add 2 ug of primary antibody (5-10 ul), incubate at 4C for 2 hours by shaking.
10. Take 30 ul of Agorose-Protein A beads into eppendorff tube and wash with 1 ml of cold PBS buffer three times, gently votex and spin 4000 rpm for 1 min.
11.  Add 50 ul of 1*SDS loading buffer and heat at 100C for 5 min and then cool down before loading on the gel.
12. Transfer the protein on the gel to the NC membrane and block the membrane.
13. Incubate with primary antibody overnight and secondary antibody (p Y: 1: 5000; Anti-IgG: 1:2000) for 1 hour.
14. ECL and develop the film.