JNK Kinase Assay
- Take
cells on 100mm Petri-style tissue culture dishes following treatment or
manipulation.
- Wash
the cells three times with PBS.
- Lyse
the cells with 1 ml lysis buffer. Incubate 30 min on ice (or in 40C
shaking).
- Syringe
and incubate 30 min on ice.
- Centrifuge
for 20 min at 40C at 15,000g.
- Check
the supernatant for protein concentration by Bradford assay.
- Take
200ug of protein solution and add ddH2O to bring the solution
to 100ul.
- Add
5ul anti-p-jnk antibody to this solution and incubate rotating overnight
at 40C.
- Add
20ul 50% Protein A-Sepharose bead slurry. Incubate rotating 1-3 hours at 40C.
- 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.
- Add
32ul kinase buffer, 4ul GST-c-jun (1ug/ul), and 4ul ATP (1mM) and incubate
at 370C for 15 min.
- 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