Yeast protein extraction, co-immunoprecipitation and Western blot analysis

Yeast cells from 100 ml liquid cultures were harvested at an OD₆₀₀ ∼ 1 and resuspended in 800 μl ice-cold B60 buffer (50 mM HEPES–KOH pH 7.3, 60 mM KAc, 5 mM Mg(Ac)₂, 0.1% Triton X-100, 10% glycerol, 1 mM NaF, 20 mM β-glycerophosphate, 1 mM DTT) supplemented with Complete protease inhibitor cocktail (Roche, Germany). Following addition of 600 μl glass beads, cell disruption was carried out at 4°C by vigorously shaking (5× for 1 min). Cell debris was removed by centrifugation at 15 000 rpm and 4°C for 5 min. An additional clearing step of the supernatant was performed at 15 000 rpm and 4°C for 20 min. Protein concentration of cleared lysates was determined using Protein Assay Dye reagent (Bio-Rad, USA). Co-immunoprecipitation was done on the cleared lysate, using either anti-c-myc (9E10, Santa Cruz Biotechnology, USA) or anti-HA (F-7, Santa Cruz Biotechnology, USA) antibodies coupled to magnetic Dynabeads (Invitrogen, USA) according to manufacturer's instructions. 2 mg lysate was incubated with 2 μg antibody-coupled Dynabeads for 60 min with end-over rotation at 4°C. Antibody bound fractions were collected using a magnetic rack and washed 3× with B60 buffer. Proteins were eluted from the beads by addition of 50 μl 1× Laemmli sample buffer (65) and incubating at 99°C for 10 min. Control samples prior to immunoprecipitation (Pre-IP) were taken from lysates before addition of antibody-coupled Dynabeads and boiled at 99°C for 10 min in 1× Laemmli sample buffer. Protein samples were separated by SDS-PAGE and analyzed by Western blotting using anti-c-myc (9E10, Thermo Fisher Scientific, USA), anti-HA (Ab-1, Dianova, Germany) and anti-Cdc19 serum (kindly provided by Dr J. Thorner, University of California, Berkley, CA, USA).