Changes

from PMID 17560372

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

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).