Difference between revisions of "Yeast Sch9 Phosphorylation Assay"

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from PMID 17560372
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==Materials==
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* NTCB dissolve to 7.5 mM in water (1.68 mg/mL)
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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
 +
# 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.
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# 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.
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# Add lysis buffer and blot using HA antibodies.
  
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TORC1 was purified from RL175-2d or RL176-1b cells treated with drug vehicle or 200 nM rapamycin for 30 min. Cells grown at 30°C in YPD (250 ml per assay point) to an OD600 of not, vert, similar1.0 were chilled on ice, collected by centrifugation, washed with H2O, resuspended in lysis buffer (1× PBS, 10% [w/v] glycerol, 0.5% [v/v] Tween 20, PI, and PPi), transferred to 2 ml screw-cap tubes half-filled with glass beads (0.5 mm), and disrupted in a Fast Prep machine at 4°C (Bio101; 5× 30 s at max. speed). Crude lysates were cleared of debris with two 1000 × g spins and protein concentrations normalized as necessary. Extracts were precleared over CL-4B Sepharose before 7 μl of IgG Sepharose (GE Healthcare) per assay point was added and the mix rotated for 90 min at 4°C. Beads were collected in a column, washed with cold lysis buffer, and aliquotted to 1.5 ml tubes. Kinase reactions were performed in kinase buffer (1× PBS, 20% glycerol, 0.5% Tween 20, 4 mM MgCl2, 10 mM DTT, 2 μg/ml heparin, and PI [−EDTA]) in a final volume of 30 μl containing not, vert, similar350 ng of recombinant Sch9. Assays were started with the addition of 100 μM ATP and 50 μCi [γ-32P]ATP, shaken for 20 min at 30°C, and terminated with the addition of 8 μl of 5× SDS-PAGE sample buffer. Samples were heated to 95°C for 5 min before being separated by SDS-PAGE, stained with Coomassie, and analyzed using a BioRad Molecular Imager.
 
  
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from PMID 19748353
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==From Park et al==
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Log phase cells were resuspended in 1 ml ice cold 0.2 M NaOH containing 0.2 % β-
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Sch9 Phosphorylation Analyses
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mercaptoethanol (v/v) and incubated on ice for 10 min. Then 50 μl of trichloroacetic acid
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was added and samples were incubated on ice for 10 min. 1M Tris base was added, and
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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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samples were boiled in 2X SDS sample buffer. SDS-PAGE and transfer to nitrocellulose
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was performed using standard protocols.  Proteins were blotted from a 6% gel and a 4-20% gel
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from PMID 18513215
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Sch9 and Ypk2 carboxy-terminal phosphorylation
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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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Latest revision as of 13:16, 24 June 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

  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.


From Park et al

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

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