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

Lyse cells (in 6 wells) with 500 µl per well of Lysis buffer (+ protease inhibitors):
20 min at 4°C.
Clear by centrifugation (14000 rpm, 4°C 15 min Eppendorf centrifuge). Save an aliquot (30 µl) for Western blotting.
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).
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.
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.
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).
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