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Kinase Assay

Kinase Assay

Lysis buffer (final conc.): for 20 ml:

20 mM Tris/HCl pH7.5

400 µl 1 M

150 mM NaCl

600 µl 5 M

25 mM b-glycerophosphate

500 µl 1 M

2 mM EDTA

80 µl 0.5 M

2 mM pyrophosphate

400 µl 0.1 M

1 mM orthovanadate

200 µl 0.1 M

1% Triton X-100

2 ml 10%

1 mM DTT

20 µl 1 M

1 mM NaF

20 µl 1 M

A. dest.

15.8 ml

 

Protease Inhibitors: added before use (Leupeptin, Pepstatin, Pefa-Block) according to stock

 

Kinase buffer (final conc.):                    for 20 ml:

20 mM Tris/HCl pH7.5

400 µl 1 M

20 mM b-glycerophosphate

400 µl 1 M

100 µM orthovanadate

20 µl 0.1 M

10 mM MgCl2

200 µl 1 M

50 mM NaCl

200 µl 5 M

1 mM DTT

20 µl 1 M

50 µM ATP

50 µl 20 mM

1 mM NaF

20 µl 1 M

A. dest.

18.7 ml

 

  1. Lyse cells (in 6 wells) with 500 µl per well of Lysis buffer (+ protease inhibitors):
    20 min at 4°C.
  2. Clear by centrifugation (14000 rpm, 4°C 15 min Eppendorf centrifuge). Save an aliquot (30 µl) for Western blotting.
  3. Immunoprecipitate the kinase (e.g. with 10 µl anti-flag affinity matrix beads, Sigma, for flag-tagged transfected kinase; or with appropriate antibody for endogenous kinase + Protein A-Sepharose or directly coupled to agarose): 2h at 4°C (rotating).
  4. Wash the beads: 3x with 1 ml PBS (4°C), 1x with 1 ml Kinase buffer (4°C): pellet the beads by centrifugation (14000 rpm, 4°C, 45sec) and remove the supernatant.
  5. Prepare Kinase buffer: add MnCl2 to 10 mM (stock: 1 M) and 32P-g-ATP (5 µCi per sample, usually 1/10 volume, i.e. 1 µl of stock solution for one 10 µl assay) and preincubate at 30°C for 10 min.
  6. Add  1 µg substrate: GST-IkB (1 µl) or mutant substrate (as control) to the beads; add 10 µl kinase buffer, mix gently and incubate at 30°C for 30 min (or longer).
  7. Stop the reaction by addition of 4x SDS-sample buffer (4 µl) and perform SDS-PAGE with the samples, followed by fixation of the gel (10% methanol, 10% HAc), drying and autoradiography.

 

For detection with PhastGel: use only 5 µl beads, 5 µl kinase buffer, 0.5 µl substrate and 2 µl 4x SDS-sample buffer: Run a 12.5% PhastGel with 4 µl per sample