Immunofluorescence Staining of Protein Complexes

After fixation, beads were washed in AbDil (20 mM Tris-HCl, pH 7.4, 150 mM NaCl with 0.1% Triton X-100, and 2% bovine serum albumin), pipetted onto poly(l-lysine)-coated coverslips and allowed to adhere for ≥30 min. Coverslips were stained in primary antibody diluted in AbDil for ≥20 min and then washed in AbDil. Primary antibodies used were 2 µg/ml FLAG (F7425 [rabbit] and F1804 [mouse], both obtained from Sigma-Aldrich), 0.25 µg/ml Myc (4A6; EMD Millipore) 1 µg/ml xCENP-C (rabbit, raised and purified against xCENP-C^207–296; Milks et al., 2009 (link)), and 1.5 µg/ml xM18BP1 (rabbit, raised against GST-xM18BP1-2 amino acids 161–415 and purified against xM18BP1-1^161–375; Moree et al., 2011 (link)). Coverslips were then stained in secondary antibodies diluted in AbDil for ≥20 min, and then washed in AbDil. Secondary antibodies used were Alexa Fluor 488–conjugated donkey anti–rabbit (Jackson ImmunoResearch Laboratories, Inc.), Alexa Fluor 647–conjugated donkey anti–rabbit (Jackson ImmunoResearch Laboratories, Inc.), and Alexa Fluor 647–conjugated goat anti–mouse (Life Technologies) all at 2 µg/ml. Coverslips were washed in PBST and PBS, gently blotted with filter paper, and mounted in 90% glycerol, 10 mM Tris, pH 8.8, 0.5% p-phenylenediamine. Coverslips were sealed to a slide with clear nail polish.