Difference between revisions of "Yeast Sch9 Phosphorylation Assay"
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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. | 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 | from PMID 19748353 | ||
| − | + | Sch9 Phosphorylation Analyses | |
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). | 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). | ||
Revision as of 16:17, 15 January 2010
from PMID 17560372
Kinase Assay
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.
from PMID 19748353
Sch9 Phosphorylation Analyses
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).
from PMID 18513215
Sch9 and Ypk2 carboxy-terminal phosphorylation
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).
