Difference between revisions of "Yeast Sch9 Phosphorylation Assay"
From Bridges Lab Protocols
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==Materials== | ==Materials== | ||
| − | * NTCB dissolve to 7.5 mM in water ( | + | * NTCB dissolve to 7.5 mM in water (1.68 mg/mL) |
* '''Urea Lysis Buffer''': 50 mM Tris [pH 7.5], 5 mM EDTA, 6 M urea, 1% SDS, 1 mM PMSF, and 0.5× PPi | * '''Urea Lysis Buffer''': 50 mM Tris [pH 7.5], 5 mM EDTA, 6 M urea, 1% SDS, 1 mM PMSF, and 0.5× PPi | ||
* '''PPi''': 10 mM NaF, 10 mM NaN3, 10 mM p-nitrophenylphosphate, 10 mM Na2P2O4, and 10 mM β-glycerophosphate; PI: 1× Roche protease inhibitor cocktail and 1 mM PMSF. | * '''PPi''': 10 mM NaF, 10 mM NaN3, 10 mM p-nitrophenylphosphate, 10 mM Na2P2O4, and 10 mM β-glycerophosphate; PI: 1× Roche protease inhibitor cocktail and 1 mM PMSF. | ||
Revision as of 17:04, 2 February 2010
Materials
- NTCB dissolve to 7.5 mM in water (1.68 mg/mL)
- Urea Lysis Buffer: 50 mM Tris [pH 7.5], 5 mM EDTA, 6 M urea, 1% SDS, 1 mM PMSF, and 0.5× PPi
- PPi: 10 mM NaF, 10 mM NaN3, 10 mM p-nitrophenylphosphate, 10 mM Na2P2O4, and 10 mM β-glycerophosphate; PI: 1× Roche protease inhibitor cocktail and 1 mM PMSF.
Protocol
- Grow cells to mid log phase
- Dilute to OD = 0.2 in YPD with or without inhibitor treatments.
- Incubate 1h at 24C in YPD
- Add TCA to a final concentration of 6% (180 uL for 3 mL culture volume)
- Place cells on ice for 5 min
- Spin cells at 5000 RPM for 5 min.
- Wash with 1 mL Acetone, spin and wash with acetone again and aspirate acetone.
- Dry cells in a speed-vac for 30 min.
- Resuspend in 150 uL of Urea Lysis Buffer.
- Lyse with a beadbeater (3x 30s).
- Heat at 65C for 10 min.
- Spin 2 min at high in eppendorf centrifuge.
- Remove 100 uL and add 30 uL of 0.5M CHES and 20 uL of NTCB. Save an untreated lysate sample as well.
- Incubate overnight at room temperature.
- Add lysis buffer and blot using HA antibodies.
