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JNK Kinase Assay

JNK Kinase Assay


Triton Lysis Buffer


20 mM Tris, PH7.4

137 mM NaCl

25 mM b-glycerophosphate

2 mM NaPPi


1 mM Na3VO4

1% Triton X-100

10% glycerol


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 assay buffer


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