JNK Kinase Assay

 

Triton Lysis Buffer

 

20 mM Tris, PH7.4

137 mM NaCl

25 mM b-glycerophosphate

2 mM NaPPi

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