Yeast Sch9 Phosphorylation Assay

• 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.

1. Grow cells to mid log phase
2. Dilute to OD = 0.2 in YPD with or without inhibitor treatments.
3. Incubate 1h at 24C in YPD
4. Add TCA to a final concentration of 6% (180 uL for 3 mL culture volume)
5. Place cells on ice for 5 min
6. Spin cells at 5000 RPM for 5 min.
7. Wash with 1 mL Acetone, spin and wash with acetone again and aspirate acetone.
8. Dry cells in a speed-vac for 30 min.
9. Resuspend in 150 uL of Urea Lysis Buffer.
10. Lyse with a beadbeater (3x 30s).
11. Heat at 65C for 10 min.
12. Spin 2 min at high in eppendorf centrifuge.
13. Remove 100 uL and add 30 uL of 0.5M CHES and 20 uL of NTCB. Save an untreated lysate sample as well.
14. Incubate overnight at room temperature.
15. Add lysis buffer and blot using HA antibodies.

Log phase cells were resuspended in 1 ml ice cold 0.2 M NaOH containing 0.2 % β- mercaptoethanol (v/v) and incubated on ice for 10 min. Then 50 μl of trichloroacetic acid was added and samples were incubated on ice for 10 min. 1M Tris base was added, and samples were boiled in 2X SDS sample buffer. SDS-PAGE and transfer to nitrocellulose was performed using standard protocols. Proteins were blotted from a 6% gel and a 4-20% gel