Native kinetochore particles were purified from asynchronously growing S. cerevisiae cells as described below. Dsn1-6His-3Flag was immunoprecipitated with anti-Flag essentially as described in Akiyoshi et al., 2010. Cells were grown in yeast peptone dextrose (YPD) rich medium. For strains containing STU2-AID, cells were treated with 500 μM auxin 30 min prior to harvesting. Protein lysates were prepared by mechanically disrupted in the presence of lysis buffer using glass beads and a beadbeater (Biospec Products). Lysed cells were resuspended in buffer H (BH) (25 mM HEPES pH 8.0), 2 mM MgCl2, 0.1 mM EDTA, 0.5 mM EGTA, 0.1% NP-40, 15% glycerol with 150 mM KCl containing protease inhibitors (at 20 μg/mL final concentration for each of leupeptin, pepstatin A, chymostatin, and 200 μM phenylmethylsulfonyl fluoride) and phosphatase inhibitors (0.1 mM Na-orthovanadate, 0.2 μM microcystin, 2 mM β-glycerophosphate, 1 mM Na pyrophosphate, 5 mM NaF) followed by centrifugation at 16,100 g for 30 min at 4°C to clarify lysate. Dynabeads conjugated with anti-Flag antibodies were incubated with extract for 3 hr with constant rotation, followed by three washes with BH containing protease inhibitors, phosphatase inhibitors, 2 mM dithiothreitol (DTT), and 150 mM KCl. Beads were further washed twice with BH containing 150 mM KCl and protease inhibitors. Associated proteins were eluted from the beads by boiling in 2× SDS sample buffer.

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