Co-immunoprecipitation for Protein Interactome

For co-immunoprecipitation (co-IP) experiments, cells were harvested at ~ 3.5-h post-meiotic induction (at the stage with maximum nuclear elongation), washed, resuspended in ice-cold Tris lysis buffer (100 mM Tris-Base, Tris-HCl, 1 M KCl, 1 M MgCl, 1% Triton X-100, 0.01 M PMSF, pH 7.5), and ground in a Dounce homogenizer. The cell lysate was clarified, filtered, and incubated with anti-HA magnetic beads (Thermo Fisher Scientific, Waltham, MA) for 2 h at 4 °C. (For details of the procedure, see Shodhan et al. 2017 (link).) After washing, two thirds of the protein-loaded beads were analyzed by mass spectrometry and protein eluted from the remaining third was analyzed by Western blotting.