Difference between revisions of "Yeast Sch9 Phosphorylation Assay"

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(modified to a protocol)
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from PMID 17560372
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==Materials==
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* NTCB dissolve to 7.5 mM in water (
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* '''Urea Lysis Buffer''': 50 mM Tris [pH 7.5], 5 mM EDTA, 6 M urea, 1% SDS, 1 mM PMSF, and 0.5× PPi
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* '''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.
  
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Kinase Assay
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==Protocol==
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# Grow cells to mid log phase
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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.
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# Dilute to OD = 0.2 in YPD with or without inhibitor treatments.
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# Incubate 1h at 24C in YPD
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Cultures were mixed with TCA (final concentration 6%) and put on ice for at least 5 min before cells were pelleted, washed twice with cold acetone, and dried in a speed-vac. Cell lysis was done in 100 μl of urea buffer (50 mM Tris [pH 7.5], 5 mM EDTA, 6 M urea, 1% SDS, 1 mM PMSF, and 0.5× PPi) with glass beads in a bead beater with subsequent heating for 10 min to 65°C. For NTCB cleavage 30 μl of 0.5 M CHES (pH 10.5) and 20 μl of NTCB (7.5 mM in H2O) were added and samples incubated over night at RT before 1 vol of 2× sample buffer (+20 mM TCEP and 0.5× PPi) was added. Further analysis was done by SDS-PAGE and immunoblotting using anti-HA antibody 12CA5 or anti-T570-P antiserum.
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# Add TCA to a final concentration of 6% (180 uL for 3 mL culture volume)
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# Place cells on ice for 5 min
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from PMID 19748353
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# Spin cells at 5000 RPM for 5 min.
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# Wash with 1 mL Acetone, spin and wash with acetone again and aspirate acetone.
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Sch9 Phosphorylation Analyses
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# Dry cells in a speed-vac for 30 min.
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# Resuspend in 150 uL of Urea Lysis Buffer.
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To analyze Sch9T570A-HA5 C-terminal phosphorylation, we used the chemical fragmentation analysis as described previously ([Urban et al., 2007] and [Wanke et al., 2008]). For quantifications of Sch9 phosphorylation, NTCB-cleaved extracts were separated by 7.5% SDS-PAGE followed by immunoblotting with anti-HA antibody 12CA5. The anti-HA antibody was detected with far-red fluorescent Alexa Fluor 680 dye-labeled secondary anti-mouse antibody (Invitrogen, A21057), and fluorescence intensity was measured using the Odyssey Infrared Imaging System (LI-COR).
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# Lyse with a beadbeater (3x 30s).
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# Heat at 65C for 10 min.
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from PMID 18513215
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# Spin 2 min at high in eppendorf centrifuge.
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# Remove 100 uL and add 30 uL of 0.5M CHES and 20 uL of NTCB. Save an untreated lysate sample as well.
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Sch9 and Ypk2 carboxy-terminal phosphorylation
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# Incubate overnight at room temperature.
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# Add lysis buffer and blot using HA antibodies.
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To analyse Sch9-5HA C-terminal phosphorylation, TB50 cells containing plasmids pJU450 and pJU676 were grown in SC-Ura, -His, -Leu to mid-log phase, harvested and re-suspended in YPAD + 0.2% Gln at 0.5 OD600. Cells were grown for 60 min at 30°C prior to addition of medium containing rapamycin or caffeine and subsequent incubation for another 30 min. Chemical fragmentation analysis was done as described (Urban et al., 2007). To analyse Ypk2 phosphorylation, MP8 cells were grown in YPD + 0.2% glutamine at 30°C to an OD600 between 0.6 and 0.8, at which point rapamycin or caffeine was added to the indicated final concentration. Cells were shaken for an additional 30 min and then harvested as described in Urban et al. (2007), but without 2-nitro-5-thiocyanobenzoic acid (NTCB) cleavage. Proteins were resolved by SDS-PAGE, transferred to nitrocellulose membrane and immunoblotted with anti-HA antibody or rabbit anti-phospho-T659 Ypk2 antiserum (this antiserum detects both Sch9 phosphorylated at T737 by TORC1 as well as Ypk2 phosphorylated at T659 by TORC2; R. Loewith, unpublished).
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Revision as of 16:53, 2 February 2010

Materials

  • NTCB dissolve to 7.5 mM in water (
  • 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

  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.
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