Na934v

Nucleosome binding tests were performed using purified HeLa mononucleosomes (Epicypher, 16–0002) resolved by 15% SDS-PAGE, transferred, and probed similar to a Western blot with modifications. For each lane, 1 μg of nucleosomes was run and after transfer, lanes were separated for individual incubation with the indicated reader domain or antibody. After blocking with 5% non-fat milk in 1 X TBST (10 mM Tris, 150 mM NaCl, 0.05% Tween-20, pH 7.6) for 15 minutes, a single lane was incubated with the indicated concentration of chromodomain in blocking buffer (or blocking buffer alone for the blots used for the commercial antibody) for one hour at room temperature with shaking. The strip blots were then washed three times for five minutes with 1 X TBST and subsequently incubated with 1:1000 anti-MBP antibody (ThermoFisher, PA1–989) or the indicated concentration of anti-H3K9me3 antibody (Active Motif, 39161) for one hour at room temperature with shaking. The blots were again washed three times for five minutes with 1 X TBST, after which they were incubated with anti-Rabbit-HRP at 1:20,000 (GE, NA934V) in blocking buffer for one hour, with shaking, at room temperature. Last, the blots were washed three times for five minutes and then imaged after incubation with a chemiluminescent substrate according to the manufacturer (GE, RPN2232).

Western blotting was done as previously described (Stice et al., 2011 (link)). The membrane was stained with Ponceau after transfer to verify the proteins were equally loaded. An internal control was used to allow comparison among different membranes. Values were normalized to the internal control to correct for variations amongst blots as previously described (Voss et al., 2003 (link)). The same mitochondrial samples were analyzed for ATP-synthase (ab109867, Abcam, Cambridge, MA), NADH dehydrogenase flavoprotein 1 (NDUFV1) (ab55535, Abcam, Cambridge, MA), Tripartite motif-containing protein 72 (TRIM72) (ab68061, Abcam, Cambridge, MA), Heat shock cognate 70 (HSC 70) (PA5–29241, Thermo Scientific, Rockford, I.L.), and Voltage- dependent anion channel-1 (VDAC-1) (ab15895, Abcam, Cambridge, MA) with 1:1000 dilution, and NADH dehydrogenase 1 alpha subcomplex subunit 5 (NDUFA5) (ab119308, Abcam, Cambridge, MA) with 1:100 dilution. Anti-mouse secondary antibody and anti-rabbit secondary antibody (respectively NA931V, NA934V GE Healthcare, Pittsburgh, PA) were used at a 1:1000 dilution.
After washing, the membrane was developed with a chemiluminescent system and analyzed as previously described (Stice, Chen, 2011 (link)).