ACTIVATION OF p70^s6k and MAP KINASES:

IP AND KINASE ACTIVITY

1. Step down and stimulate cells as required. 5 minutes usually represents the maximum of MAP kinase activation following insulin stimulation, with activity close to zero by 15 minutes. For p70^s6k 30 minutes is a good time for near maximal. Time course of activation is slower than for MAPK and stays up for about 2 hours in 32-D cells. Wash cells and lyse:

a. suspension cells: dilute cells with several volumes of ice-cold STE and collect by centrifugation (cold). Add 1 ml lysis buffer, resuspend cell pellet with pipetter and transfer to eppendorf. Allow lysis to contiue 5-10 minutes at 4^o C.

b. adherent cells: shake off media, place cells on ice and wash twice (gently) with ice-cold STE. Shake off wash and add 1 ml lysis buffer per 10 cm plate. Allow to lyse for 10 minutes on 4^o C rocker, scrape cells and transfer to eppendorf.

2. Spin crap out of lysate in cold microfuge, 10 minutes. Transfer supernatant to new tube. Perform protein assay if necessary. IP 1/4-1/2 of lysate (adjusted to protein concentration, if necessary) (use entire sample or >50% of sample if from 32-D cells) per point, using 5 ml of ap70^s6k or aMAPK serum (from Blenis). Incubate at 4^o C for 1-2 hours. Add 50 ml of BSA-blocked protein A sepharose 6MB (1:1 slurry with PBS), put on rotator and incubate at 4^o C for at least one hour. NOTE: you will need to cut off the end of your pipette tip so that the sepharose beads will enter your pipette tip when adding it. Also, the beads settle quickly, so agitate the stock frequently when adding it to your samples, so that yu get equivalent amounts in each tube.

3. Wash IP's 2 times with wash A, 2 times with wash B, once with ST. Aspirate last wash as completely as possible.

4. Add 20 ml/tube 1.5x kinase buffer.

5. Add 10 ml/tube of start buffer, staggering starts 20", so that each reaction can be stopped after incubating exactly 15'. After 15', stop reactions every 20" by adding 40 ml 2x LSB+DTT and boiling for 5'.

6. Load laemli-ized samples onto 12.5% SDS gel (large gel) and electrophorese until dye front runs off of bottom. Do not run too far, as MBP runs at about 20 kDa.

7. Stain/Destain gel. Soak gel in 10% glycerol for at least 1 hour before drying (high percentage gels tend to crack during drying process otherwise). Dry gel on dryer with strong pump, low heat (50 C) for 3-4 hours. Expose to film or phosphorimager.

BUFFERS:

ST

NaCl 150 mM

Tris pH 7.2 50 mM

STE

NaCl 150 mM

Tris pH 7.2 50 mM

EDTA 1 mM

LYSIS BUFFER

NP-40 0.5%

KPO_{4/EDTA 10 mM/ 1mM}

_{(from 10X stock: 100 mM KPO4/10 mM EDTA pH 7.05)}

_{EGTA 5 mM}

_{MgCl2 10 mM}

_{DTT 2 mM}

_{b-glycerophosphate 50 mM}

_{(from 1 M stock pH 7.2)}

_{Na3VO4 1 mM}

_{*PMSF 40 mg/ml}

_{*Aprotinin, Leupeptin 10 mg/ml each}

_{BUFFER A}

_{NP-40 1%}

_{Deoxycholate 0.5%}

_{NaCl 100 mM}

_{Tris pH 7.2 10 mM}

_{EDTA 1 mM}

_{Na3VO4 1 mM}

_{DTT 2 mM}

_{*PMSF 40 mg/ml}

_{BUFFER B}

_{NaCl 1 M}

_{NP-40 0.1%}

_{Tris pH 7.2 10 mM}

_{DTT 2 mM}

_{*Na3VO4 1 mM}

*PMSF 40 mg/ml

10X KINASE BUFFER (note: make this as stock and dilute some to 1.5X. Store both frozen, thawing aliquots of 1.5X as needed)

Hepes pH 7.2 200 mM

MgCl_{2 100 mM}

_{b-mercaptoethanol 30 mM}

_{BSA 10 mg/ml}

_START

_{cold ATP 50 mM final}

_{hot ATP 20 mCi/rxn.}

_{SUBSTRATE: MAP kinase: MBP 2 mg/pt}

_p70^s6k: 40S ribosomes 20 mg/pt.

*ADD FRESH