Affinity Purification of Protein Complexes

Yeast cells expressing the indicated fusion proteins were harvested by filtration, frozen in liquid nitrogen, and cryogenically disrupted using the Precellys homogenizer in 4 mL of lysis buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.1% NP-40, 10% glycerol, 400 mM Pefabloc, and Roche complete protease inhibitor EDTA-free) with the addition of 60 mM glutamine as in Ukai et al., 2018 (link). Cleared lysates were equilibrated in the same lysis buffer. For input samples, aliquots of cleared lysates were collected and denatured in presence of SDS-PAGE sample buffer. For co-immunoprecipitations, the cleared lysates were incubated for 2 h at 4 °C with prewashed anti-c-myc MagBeads (Pierce Thermo Fisher Scientific, product number 88843). After five washes with lysis buffer, beads were resuspended in 20 µL lysis buffer and denatured in presence of SDS-PAGE sample buffer. Inputs and pull-down samples were analyzed by SDS-PAGE immunoblot with mouse anti-myc (Santa Cruz, 1:10,000) and mouse anti-HA (ENZO, 1:1000) antibodies.