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