Kinase assay (T-associated kinase reaction)

Kinase assay (T-associated kinase reaction)
1. Gently collect the cells in PBS (especially if cells were treated with Nocodazole) and
centrifuge 1200 rpm 5 min. Aspirate supernatant and extract the cell pellet with 1 ml
TEB supplemented with 2 mM EDTA, PMSF, leupeptin, pepstatin, 0.5 mM Na3VO4,
10 mM NaF, 10 mM -glycerophosphate for 15 min. on ice.
2. Centrifuge at 10,000xg for 10 min and transfer lysate to new tube.
3. Add the appropriate antibody for immunoprecipitation (e.g. SV40 T antibody
PAb419) and incubate on mixer at 4 ˚C for 2 hrs
4. Collect the antibody complexes by addition of 30 µl protein A sepharose beads.
Incubate another 45 min. with mixing at 4˚C.
5. Wash the beads sequentially two times with TEB and two times with kinase buffer –
make fresh, MnCl2 solution goes bad [50 mM Tris pH7.5, 5 mM MgCl2, 5 mM
MnCl2, 1 mM NaF, 10 mM -glycerophosphate, 1 mM DTT]
6. For each kinase reaction add 30 µl kinase buffer supplemented with 5 µCi [32P]ATP
(we’ll try to do the kinase reaction without adding any cold ATP in order to increase
the signal). E.g for 10 reactions make up enough for 11 samples: 330 µl kinase buffer
+ 8 µl [32P]ATP (given it’s not entirely fresh)