ABSTRACT: Kinetochores were purified (via Dsn1-His-Flag) from Saccharomyces cerevisiae lysate from a wildtype background (CN091523_101423_SBY8253, CN040221_042921_SBY8253, CN020524_020824_SBY8253), an mps1-1 genetic background grown at 37 C for 2.5 hours (CN091523_101423_SBY8726, CN020524_020824_SBY8726) and a pGAL-MPS1 background grown in 4% galactose for 2.5 hours (CN020524_020824_SBY8810, CN040221_042921_SBY8810). The Ndc80c (Ndc80-Flag) was purified from S. cerevisiae lysate from a wildtype (CN030221_031021_10651, CN020524_020824_SBY10651) or pGAL-MPS1 genetic background (CN030221_031021_19721, CN020524_020824_SBY19721) grown in 4% galactose for 2.5 hours prior to harvest. The Dam1c (Dad1-Flag) was purified from S. cerevisiae lysate from a wildtype (CN052721_053021_12441, CN020524_020824_SBY12441) or pGAL-MPS1 genetic background (CN052721_053021_20761) grown in 4% galactose for 2.5 hours prior to harvest. Kinetochores were purified (Dsn1-His-Flag) from NDC80WT cells (CN032723_033023_SBY22006, CN051322_052422_SBY21353), ndc80T248A,T252A cells (CN032723_033023_SBY22007, CN051322_052422_SBY21287) and ndc80T248D,T252D cells (CN032723_033023_SBY22008, CN051322_052422_SBY21289). Kinetochores were purified (Dsn1-His-Flag) from NDC80WT (CN032723_033023_SBY22062), ndc80T248A,T252A (CN032723_033023_SBY22063) and ndc80T248D,T252D cells (CN032723_033023_SBY22011), all cdc15-2 (anaphase arrested) at 37 C for 2.5 hours prior to harvest. Harvested yeast were resuspended in Buffer H (25 mM HEPES pH 8.0, 150 mM KCl, 2 mM MgCl2, 0.1 mM EDTA pH 8.0, 0.1% NP-40, 15% glycerol) supplemented with protease and phosphatase inhibitors. After resuspension and re-spinning, yeast pellets were frozen in liquid nitrogen and lysed using a Freezer Mill (SPEX, Metuchen NJ). Lysate was clarified via ultracentrifugation at 24,000 RPM (98,000 x g) for 90 minutes and the protein layer was extracted with a syringe. This extract was incubated with magnetic a-M3DK antibody conjugated Dynabeads (Invirtrogen, Waltham MA) for 3 hours at 4 C with rotation. Dynabeads were washed with 10x bead volume of Buffer H 5 times (the last 3 washes omitting DTT and phosphatase inhibitors). For mass spectrometry, kinetochores were eluted from Dynabeads with 0.2% RapiGest (Waters Corporation, Milford MA) in 50 mM HEPES pH 8.0.
Susan Biggins 
