Immunoprecipitation and Protein Detection

As previously described (33 (link)), the indicated strains were cultured in 50 ml YEAU and harvested at log phase. Cell pellets were then washed and cryogenically disrupted with FastPrep MP with two pulses (60 sec) of bead-beating in ice-cold lysis buffer (50 mM HEPES at pH 7.5, 140 mM NaCl, 15 mM EGTA, 15 mM MgCl₂, 0.1% NP40, 0.5 mM Na₃VO₄, 1 mM NaF, 2 mM PMSF, 2 mM benzamidine, Complete proteinase inhibitor [Roche]). After clearing by centrifugation, protein concentrations were measured via Bradford assay and adjusted to 12 mg/ml. Anti-Flag M2 affinity gel (Sigma), anti-Myc (9E10 from Santa Cruz Biotechnology) or anti-Ccq1 rabbit serum plus IgG beads (Roche) was used for immunoprecipitation, followed by eluting with 30 μl 0.1 M glycine (pH 2.0) at room temperature for 10 min. The elute was immediately neutralized with 2 μl 2 M Tris–HCl, pH 8.0. SDS-PAGE (8%) and western blotting using monoclonal anti-Flag (M2-F1804, from Sigma), monoclonal anti-Myc (from Covance), monoclonal anti-PK (ab27671 from Abcam), anti-Ccq1 rabbit serum (16 (link)), or anti-Cdc2 (y100.4, from Abcam) were performed to detect protein–protein interaction as indicated.