JNK Kinase Assay

  1. Take cells on 100mm Petri-style tissue culture dishes following treatment or manipulation.
  2. Wash the cells three times with PBS.
  3. Lyse the cells with 1 ml lysis buffer. Incubate 30 min on ice (or in 40C shaking).
  4. Syringe and incubate 30 min on ice.
  5. Centrifuge for 20 min at 40C at 15,000g.
  6. Check the supernatant for protein concentration by Bradford assay.
  7. Take 200ug of protein solution and add ddH2O to bring the solution to 100ul.
  8. Add 5ul anti-p-jnk antibody to this solution and incubate rotating overnight at 40C.
  9. Add 20ul 50% Protein A-Sepharose bead slurry. Incubate rotating 1-3 hours at 40C.
  10. Centrifuge the solution at 16,000g for 5 min, save the beads and wash them rotating 2x5 min with 500ul lysis buffer and 2x5 min with 500ul kinase buffer.
  11. Add 32ul kinase buffer, 4ul GST-c-jun (1ug/ul), and 4ul ATP (1mM) and incubate at 370C for 15 min.
  12. Add 40ul of 2xSDS sample buffer and boil 3 min. Run a SDS Gel and blot for western.

Kinase Buffer:

20mM HEPES (pH 7.6)

10mM MgCl2

Autoclaved and the following added:

10mM NaF

20mM b-Glycerophosphate

Before using the solution add the following:

1:200 Na3VO4 (200mM stock-activated)

Lysis Buffer

25mM HEPES (pH 7.6)

300mM NaCl

1.5mM MgCl2

0.2mM EDTA

Autoclaved and the following added:

10mM NaF

20mM b-Glycerophosphate

0.1% Triton-X-100

Before using the solution add the following:

1:200 Na3VO4 (200mM stock-activated)

1:1000 protease inhibitors

1:1000 DTT

Protease Inhibitor Cocktail:

100mM PMSF

1mg/ml Leupeptin

1mg/ml Pepstatin A

100mM Benzamidine