Visualizing Histone Modifications by Western Blot

Western blots to visualize Hda1 and H2Bub were performed using whole-cell extracts prepared using SUTEB buffer as described (Van Oss et al. 2016 (link)). H3 methylation was examined using whole-cell extracts prepared using a TCA extraction method (Van Oss et al. 2016 (link)). Proteins were run on 15% SDS-polyacrylamide gels. The following antibodies were used: α-Hda1 (Santa Cruz sc-393814; 1:200), α-H2B (Active Motif #39237; 1:3000), α-H2Bub (Cell Signaling #5546; 1:1000), α-G6PDH (Sigma #A9521; 1:20000), α-H3K4Me2 (Millipore #07-030; 1:2000), α-H3K4Me3 (Active Motif #39159; 1:2000), α-H3K79Me2/3 (Abcam #ab2621; 1:1000; note that this antibody recognizes both di- and trimethylated H3K79) and α-H3 antibody (Tomson et al. 2011 (link)). Blots were developed using Thermo Scientific West Pico Plus (34580) and images were collected on a BioRad ChemiDoc™ XRS+.