37C for 1 hr. Finally, 50 ml of 40 mg/ml proteinase K + 1 mM SYTOX Green + 50 mM Tris pH 7.4 was added to each sample and incubated at 55C for 1 hr prior to analysis by cytometry. Measurement of meiotic divisions and sporulation efficiency Progression of meiotic divisions was measured by epifluorescence of SYTOX Green-stained cells. 100200 cells were counted per sample. To measure sporulation efficiency, we counted the proportion of tetrad, dyad, and monad or unsporulated cells from synchronous sporulation cultures after 2448 hr. Protein purification W303 S. cerevisiae strains containing a 2 mm PGAL1-kinase-TAP plasmid were grown overnight to log phase in SC-URA media containing 2% raffinose, and then expression of N-terminal kinase domains was induced by addition of 2% galactose for 4 hr at 30C. Cells were harvested by centrifugation at 8000 rpm, cell pellet washed and LOXO 101 price resuspended in 1 cell volume of lysis buffer containing 25 mM HEPES pH 8.0, 300 mM NaCl, 0.1% NP-40, 30 mM EGTA, 1 mM EDTA, and a protease/phosphatase inhibitor set was added immediately prior to harvest including 80 mM b-glycerophosphate, 50 mM NaF, 1 mM DTT, 1 mM Na3O4V, and 1 mM PMSF. The cell slurry was slowly dripped into liquid nitrogen to produce frozen pellets. These pellets were then pulverized in a cryogenic ball mill by five rounds of agitation at 15 Hz for 2 min, re-cooling the grinding jars in liquid N2 after each cycle. The grindate was then thawed and cell debris was cleared by centrifugation at 8000 rpm for 30 min followed by sequential filtration through 2.7 and 1.6 mm Whatman GD/X filters. C-terminally TAP-tagged kinases were immobilized on IgG Sepharose 6 Fast Flow beads. These beads had been pre-equilibrated in lysis buffer with inhibitors and were then incubated with lysate for 1 hr at 4C. Bound beads were then loaded into a disposable Bio-Spin column by pipette and washed with 20 ml total wash buffer at 4C. The column was then rotated for 20 min at 23C in 700 ml wash buffer as a final wash to mimic elution conditions. The bound protein was then cleaved from the IgG beads by TEV protease in 600 ml elution buffer for 1 hr at 23C. For bacterial purification, proteins were expressed with an N-terminal 6 HIS tag in Rosetta2 DE3 pLysS competent cells by induction with 100 mM IPTG for 18 hr at 16C. The cell pellet was resuspended in an equal volume of bacterial wash buffer, freeze-thawed once with liquid nitrogen, then lysed by 3 french press cycles in the presence of 0.05 mg/ml DNAse I, 2 mM MgCl2, 5 mM b-mercaptoethanol, and 1 mM PMSF. Lysate was cleared by centrifugation and filtration as above. NiNTA-Sepharose beads were equilibrated in bacterial wash buffer containing 20 mM imidazole and combined at 1:1 ratio with lysate before adding a further 2.5 mM bME, 1 mM Na3O4V, 80 mM b-glycerophosphate, 50 mM NaF, and 0.5 mM PMSF. This lysate was incubated with beads for batch binding overnight at 4C. Bound beads were loaded onto a disposable column and rinsed with the remaining unbound fraction. The column was washed 3 with 10 ml bacterial wash buffer containing 10 mM imidazole and 2 mM bME, 3 with 10 ml bacterial wash buffer containing 20 mM imidazole and 2 mM bME, PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19826048 then one final time with 20 ml bacterial wash buffer containing 20 mM imidazole, 
2 mM bME, and 2 mM ATP. The column was rotated in previous ATP solution for 15 min at 4C in an attempt to remove chaperones, before rinsing once more with 5 ml bacterial wash buffer containing 20 mM imidazole and 2 mM