In Vitro MAP kinase assay 9/1998

1. Deprive PC12 cells overnight in 4ml of DMEM+1% BSA+1%Glu/AA
2. stimulate cells with 100ng/ml for 10 minutes
3. Lyse cells in 600ml lysis buffer:
1. 50mM HEPES, pH 7.5
2. 150mM NaCL
3. 1.5mM MgCl2
4. 1mM EGTA
5. 0.1% Triton X-100
6. 105 glycerol
7. 1mM Na3VO4,1mM PMSF
8. 10mg/ml aprotinin
9. 10mg/ml leupeptin
4. Immunoprecipitate with ant-ERK2 (Santa Cruz, SC-154, 1:100 dilution, 0.5mg anti- ERK2 per condition)
5. Wash 3x1ml lysis buffer, 2x 0.5ml kinase buffer (50mM HEPES, pH 7.4, 10mM MgCl2, 0.5mM DTT, 5mM Na3VO4)
1. HEPES (50mM pH 7.4) 10ml
2. MgCl2 (2M) 50ml
3. DTT (1M) 5ml
4. Na3VO4 500ml
6. Add 10-20mg GST-SH2-Bb to anti-ERK2 immunoprecipitates (could be reduced to 1mg/reaction)
7. Add 50ml reaction buffer, mix at room temp for 30 minutes (mix every 10 min)

Reaction buffer:

Kinase Buffer 700ml

Cold ATP 3ml ([final]=20-100mM)(not necessary)

PI 1ml([final]=10mM)

g-³²PATP (150mCi/ml) 1.3ml (10mCi per reaction)

8. Add 400ml lysis buffer and mix, spin down, transfer the supernatant to a fresh tube
9. add 50ml glutathione-agarose beads, rotate in cold room for 1-2 hrs
10. wash beads with 2x 1ml lysis buffer, 2x 0.5 ml thrombin cleavage buffer (TCB:50mM Tris-HCl, pH 8.0, 150mM NaCl, 2.5mM CaCl2, 0.1%b-ME)
1. Tris-HCl, pH 8.0 10ml
2. NaCl 300ml
3. CaCl2 25ml
4. B-ME 10ml
11. Add 110 ml TCB containing 1U thrombin (Sigma, T3010), rotate at Room temp 1 hr
12. Add 30ml 5x loading buffer, boil 5 minutes, run 7.5% SDS-PAGE
13. transfer proteins to nitrocellulose membrane
14. autoradiography for 1-3 days
15. immunoblot the membrane with anti-SH2-B