(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)
|Histone Wash Buffer
|Histone H1, 2 µg/µL
|ATP, 500 µM
|32P-gamma ATP, 10 µCi/µL
RIPA cell lysis buffer
10 mM NaPO4, pH7.2
0.3 M NaCl
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 - where should this link to?