Protein detection by immunoblot and antibodies

Yeast cultures were grown to early mid-log (OD_600nm 0.5), washed in MilliQ, pelleted and snap-frozen on dry ice. For protein extraction, cells were resuspended in 300 μl MilliQ, incubated with 300 μl 0.2 M NaOH for 7 min at room temperature, centrifuged and resuspended in 500 μl 2× SDS-PAGE sample buffer (4% SDS, 20% glycerol, 0.1 M dithiothreitol (DTT), 0.125 M Tris-HCl pH 7.5 and EDTA-free protease inhibitors). Samples were incubated at 95 °C for 5 min while shaking and centrifuged at 800g for 10 min at 4 °C. Then 20 μl of lysate with loading buffer was run on a NuPAGE 3–8% gradient TAC gel (V5) or 16% polyacrylamide gel (histone H3 and H3K79me3), and transferred to a 0.45 μm nitrocellulose membrane for 4 h (V5) and 2 h (histone H3 and H3K79me3). For blocking, the membrane was washed with tris-buffered saline-tween (TBS-T), incubated with PBS containing 5% milk for 1 h at room temperature and washed briefly with TBS-T. The membrane was incubated with PBS containing 2% milk and primary antibody (1:2,000 for αV5, 1:5,000 for αPgk1 and 1:1,000 for αH3 and αH3K79me3) overnight at 4 °C, washed 3× for 10 min with TBS-T, incubated with 2% mild and secondary antibody (1:5000) for 1 h at room temperature, washed 3× for 10 min with TBS-T and 1× for 10 min with PBS, and imaged using an LI-COR Odyssey infrared imager (Biosciences). Western blot analysis was performed using antibodies against V5 (Thermo Fisher Scientific R960-25, RRID: AB_2556564), Pgk1 (Invitrogen 22C5D8, RRID: AB_2532235), histone H3 (RRID:AB_2631108, a kind gift of the F.v.L. laboratory)⁶⁵ and histone H3K79me3 (RRID: AB_2631107, a kind gift of the F.v.L. laboratory)⁶⁵. Secondary antibodies used were IRDye 800CW Goat anti-Mouse IgG 925-32210 Li-COR (RRID AB_2687825), IRDye 800CW Goat anti-Rabbit IgG 926-32211 Li-COR (RRID:AB_621843) and IRDye 680RD Donkey anti-Mouse IgG 925-68072 Li-COR (RRID AB_2814912).