This is a cached page for the URL (http://www.whitelabs.org/Lab%20Protocols/kinase%20and%20phosphatase%20assays/JNK%20Kinase%20Assay.htm). To see the most recent version of this page, please click here.
Protocol Online is not affiliated with the authors of this page nor responsible for its content.
About Cache
JNK Kinase Assay

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