HISTONE KINASE ASSAY
(M. Fero) 9/1/04
- To 1.5 mL eppendorf tubes add:
200 µg of protein extract (see Western blot protocol for protein sample preps)
q.s. to 300 µL with RIPA (with protease and phosphatase inhibitors).
Primary antibody (amount determined emperically, approx 10x the concentration needed for westerns)
- Vortex and incubate on ice for 30 min.
- Meanwhile wash Protein A-Sepharose beads 2x in RIPA as follows:
- Cut off ends of pipetman tips
- Suspend beads and add beads to a 1.5 mL tube (40µL x # of reactions)
- Spin down beads, aspirate supernatant, resuspend in 1 mL of RIPA.
- Resuspend beads in 1 vol. of RIPA (40µL x # of reactions)
- Vortex beads before adding 30 µL to each reaction (a lot is lost on tips).
- Rotate in cold room for 1 hr.
- Spin and wash beads twice with RIPA (discard supernatant), and then once with Histone Wash Buffer.
- Add 30 µL of Histone Assay solution. Incubate at 37°C for exactly 30 min.
Mix by vortexing every 10 min.
- Stop reaction by adding 15 µL of 4x sample buffer. Heat on 95°C block for 5 min.
(Can be stored at 4°C overnight)
- Spin down beads and load supernatant on a 12% SDS acrylamide gel.
Run off the dye front (175 volts x 60 min. for a BioRad MiniProtean 3 gel)
- Rinse gel in water. Stain with Coumassie x15 min. Destain for 1-2 hrs.
(This will fix the proteins into the gel).
- Wrap in Saran and expose to X-ray film at -80°C with intensifying screens. The optimal exposure time will depend on the amount of 32P used and the amount of kinase activity. Generally a few hours should be fine.
- Histone Wash Buffer
- 25 mM Tris HCl, pH7.5
- 70 mM NaCl
- 10 mM MgCl2
- 1 mM DTT
|Histone Assay Solution (10 rxns) ||Amount ||[Final] |
|Histone Wash Buffer ||320 µL ||- |
|Histone H1, 2 µg/µL ||18 µl ||0.1 µg/µL |
|ATP, 500 µM ||7 µL ||10 µM |
|32P-gamma ATP, 10 µCi/µL ||7 µL ||0.2 µCi/µL |
|RIPA cell lysis buffer |
|10 mM NaPO4, pH7.2 |
|0.3 M NaCl |
|0.1% SDS |
|1% NP40 (Nonidet P40) |
|1% DOC (sodium deoxycholate) |
|2 mM EDTA |
Protease Inhibitors (add fresh to RIPA)
Leupeptin 5 mg/mL in PBS (1000-10,000x, aliquot and store -20°C), Roche # 11 017 101 001
Aprotinin 10 mg/mL in PBS (5000-160,000x, aliquot and store -20°C), Roche # 10 981 532 001
PMSF: (phenylmethylsulfonyl flouride, 100x) 0.2 M in ethanol. Store at 4°C.
100x Phosphatase inhibitor mix (store -20ºC and add fresh to RIPA)
100 mM NaF
50 mM NaVanadate
800 mM ß-glycerol phosphate
5x Sample Buffer