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Kinase Assay

Kinase Assay

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

20 mM Tris/HCl pH7.5

400 l 1 M

150 mM NaCl

600 l 5 M

25 mM b-glycerophosphate

500 l 1 M

2 mM EDTA

80 l 0.5 M

2 mM pyrophosphate

400 l 0.1 M

1 mM orthovanadate

200 l 0.1 M

1% Triton X-100

2 ml 10%

1 mM DTT

20 l 1 M

1 mM NaF

20 l 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 l 1 M

20 mM b-glycerophosphate

400 l 1 M

100 M orthovanadate

20 l 0.1 M

10 mM MgCl2

200 l 1 M

50 mM NaCl

200 l 5 M

1 mM DTT

20 l 1 M

50 M ATP

50 l 20 mM

1 mM NaF

20 l 1 M

A. dest.

18.7 ml

 

  1. Lyse cells (in 6 wells) with 500 l per well of Lysis buffer (+ protease inhibitors):
    20 min at 4C.
  2. Clear by centrifugation (14000 rpm, 4C 15 min Eppendorf centrifuge). Save an aliquot (30 l) for Western blotting.
  3. Immunoprecipitate the kinase (e.g. with 10 l 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 4C (rotating).
  4. Wash the beads: 3x with 1 ml PBS (4C), 1x with 1 ml Kinase buffer (4C): pellet the beads by centrifugation (14000 rpm, 4C, 45sec) and remove the supernatant.
  5. Prepare Kinase buffer: add MnCl2 to 10 mM (stock: 1 M) and 32P-g-ATP (5 Ci per sample, usually 1/10 volume, i.e. 1 l of stock solution for one 10 l assay) and preincubate at 30C for 10 min.
  6. Add 1 g substrate: GST-IkB (1 l) or mutant substrate (as control) to the beads; add 10 l kinase buffer, mix gently and incubate at 30C for 30 min (or longer).
  7. Stop the reaction by addition of 4x SDS-sample buffer (4 l) 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 l beads, 5 l kinase buffer, 0.5 l substrate and 2 l 4x SDS-sample buffer: Run a 12.5% PhastGel with 4 l per sample