Difference between revisions of "Yeast Sch9 Phosphorylation Assay"
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Kinase Assay | Kinase Assay | ||
| − | + | 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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| + | 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. | ||
from PMID 19748353 | from PMID 19748353 | ||
Revision as of 16:22, 15 January 2010
from PMID 17560372
Kinase Assay
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.
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.
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).
