- Culture cells (MEFs etc) in plate with 10 cm in
diameter, 37C, 5% CO2 incubator.
- Starve the cells overnight at least 18 hours with
medium containing no serum.
- Treat the cells with insulin (100 nm) or others for a
time period of 30 min.
- Wash the cells with cold PBS twice.
- Add the cell lysis buffer 900 ul.
- Place the cells at 4C cold room for shaking for 30
- Harvest the cells into 1.5 ml eppendof tubes.
- Centrifuge the tubes at 14,000g for 10 min at 4 C.
- 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.
- 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.
- Add 50 ul of 1*SDS loading buffer and heat at 100C
for 5 min and then cool down before loading on the gel.
- Transfer the protein on the gel to the NC membrane and
block the membrane.
- Incubate with primary antibody overnight and secondary
antibody (p Y: 1: 5000; Anti-IgG: 1:2000) for 1 hour.
- ECL and develop the film.