JNK Kinase Assay

Triton Lysis Buffer

20 mM Tris, PH7.4

137 mM NaCl

25 mM b-glycerophosphate

2 mM NaPP_i

2 mM EDTA

1 mM Na₃VO₄

1% Triton X-100

10% glycerol

1 mM PMSF

5 mg/ml leupeptin

5 mg/ml aprotinin

2 mM benzamidine

0.5 mM DTT

Kinase Assay Buffer

25 mM Hepes, pH 7.4

25 mM b-glycerophosphate

25 mM MgCL₂

0.1 mM Na₃VO₄

0.5 mM DTT

Kinase Assay

Kinase

Kinase assay buffer

Substrate

10 mCi g-P³²- ATP

20 mM ATP

- in 50 ml

- 15 min at RT

- Stop with Sample buffer

Preparation of kinase

1. Transiently express kinase in cells – 24-36 hrs. Starve for last 12 hrs.

2. Assay after stimulation of cells with 5.0 mg/ml anisomycin/40 mM HCl

3. IP kinase with M2 antibody – 3-4 hrs

4. Elute kinase from IP with Flag peptide in Kinase Buffer (100 mg/ml) – O/N @ 4⁰C