30ug of mESC nuclear protein extracts were run on 10% Bis-Tris gels (Invitrogen NP0316BOX) in MOPS buffer (Invitrogen NP0001) at 75 volts for 20 minutes followed by 120 volts for 1 hour. Gels were incubated for 30 minutes in 20% methanol transfer buffer (Invitrogen NP0006-1) and transferred onto PVDF membranes (Invitrogen 831605) at 20 volts for 1 hour using the Bio-Rad semi-dry transfer system (170–3940). Membranes were blocked in Blotto (Pierce, 37530) at room temperature for 1 hour. Antibodies were diluted in Blotto and membranes were incubated overnight at 4°C. Antibodies were diluted in using the following concentrations. Ezh2 1:2000, Suz12 1:5000, hnRNPH 1:1000, Ruvbl2 1:1000, Jarid1b 1:500, HDAC1 1:250, Cbx6 1:500, YY1 1:500. All antibodies tested were raised in rabbit. The next day, membranes were washed 3x in 0.1% TBST for 5 minutes each. The membranes were probed with anti-Rabbit-horse radish peroxidase (GE Healthcare; NA9340V) at a 1:10,000 dilution, washed 3x in 0.1% TBST, incubated in ECL reagent (GE Healthcare RPN2132), and exposed.
