1.  Grow cells of interest to 80% confluence in 150 mm dishes.  Serum starve overnight in media containing 0.5% BSA (insulin-free), no serum.


2.  Take cells to the hot room.  Discard lids from plates and remove media, wash once with phosphate-free media, move cells into the hot box and add 5 ml of phosphate-free media containing 1-10 mCi 32P-orthophosphate (NEN cat # NEX 053) per plate.  Place cells in a lucite box and place in incubator 37oC, 5% CO2 in hot room on rocker for 2-3 hrs to label to equilibrium.


3.  Stimulate cells for the desired time with the appropriate concentration of the desired growth factor (e.g. 1 min with 50 ul of 10-5 M insulin (for 10-7 M final)).


4.  Move cells to the hot box.  Aspirate media quickly (hot waste), freeze cells by dousing in liquid nitrogen.  Pour off excess LN2, add 2 ml/plate of solubilization buffer:

                                                                                    50 ml:

            50 mM HEPES pH 7.4

            1.0% Triton X-100                                           500 ul

            2 mM Vanadate                                               18 mg

            2 mM PMSF                                                    500 ul of stock (35 mg/ml EtOH)

                       NaF                                                      210 mg

            5 mM EDTA                                                    500 ul of 0.5 M stock

5.  Thaw cells into buffer, scrape with cell lifter, transfer to polycarbonate centrifuge tubes with caps and spin in Ti70 rotor 55 krpm for 1 hour.


6.  Immunoprecipitate supernatant with (20ul) of desired antibody (2 hr-overnight), collect on 100 ul pansorbin 1+ hours.  Re-immunoprecipitate if desired.


7.  Collect pellets--  spin down, aspirate supernatant (hot waste).  Add 1 ml Tris-SDS IP wash, sonicate.  Repeat 2 additional times.  Aspirate supernatant, add 70 ul of laemmli sample buffer.


8.  Boil 5', vortex to resuspend pellet, boil 3'.  Spin down pansorbin 5', load supernatant to gel.