Immunoprecipitation and Mass Spectrometry

Immunoprecipitation was performed using 200 μg of protein and using Pierce MS-Compatible Magnetic IP Kit, protein A/G (90409, Thermo Fisher Scientific) following manufacturer instructions in SDS-free conditions. Protein extracts were pulled down using 10 μg BMI1 antibody (39993, Actif Motif) or 10 μg RYBP antibody (AB3637, Millipore). Protein eluates diluted in 40 μl of 100 mM AMBIC buffer (Sigma) were analysed with MS. Extensively validated antibodies were used (45–48 (link)). For IP experiments, unbound samples were analysed to confirm sensitivity of the beads. For MS experiments proteins unspecifically binding to beads were subtracted by comparing BMI1-IP samples with mock-IP samples without antibody for each condition.