Rabbit anti acetyl histone h3

Log-phase cells (∼30 ml) were harvested by centrifuging at 5000 ×g for 5 min. The pellet was resuspended in 100 µl Laemmli sample buffer (Bio-Rad 1610737) and heated to 95°C for 10 min. The lysate was quick-spun and the supernatant was saved. Proteins were separated by SDS–PAGE in a 15% gel and transferred to polyvinylidene difluoride membrane. All blocking and antibody incubations were performed in Tris-buffered saline containing 0.5% Tween-20 and 2% (wt/vol) bovine serum albumin (BSA). The primary antibodies were rabbit anti-histone H3 (Abcam 1791), rabbit anti-acetyl-histone H3 (Millipore 06-599), and rabbit anti-acetyl-histone H4 (Millipore 06-866). The secondary antibodies were HRP-conjugated goat-anti-rabbit immunoglobulin G (CST 7074S). The blot membrane was treated with 1 ml Clarity Western ECL substrates (Bio-Rad 1705060) and scanned with a Thermo Scientific myECL Imager.

Cells were lysed in Laemmli sample buffer supplemented with 10% 2-mercaptoethanol (133-1457; Wako) and incubated at 95°C for 5 min to denature proteins. The cell lysates, equivalent to 1 × 10⁵ cells per well, were subjected to SDS–polyacrylamide gel electrophoresis (12.5% for histone detection; 10% for RAD21 detection). For Western blotting, the fractionated proteins in the gel were transferred to a polyvinylidene difluoride (PVDF) membrane (IPVH00010; Millipore) by a semi-dry blotter (BE-320; BIO CRAFT). After blocking with 5% skim milk (Morinaga), the membrane-bound proteins were probed by the rabbit anti–acetyl histone H4 (06-866; Millipore), rabbit anti–acetyl histone H3 (06-599; Millipore), rabbit anti-H2B (ab1790; Abcam), mouse anti-RAD21 (05-908; Millipore) or goat anti–lamin A/C (sc-6215; Santa Cruz Biotechnology) antibody, followed by the appropriate secondary antibody: anti-rabbit (170-6515; Bio-Rad), anti-mouse (170-6516; Bio-Rad), or anti-goat (305-035-003; Jackson ImmunoResearch) horseradish peroxidase–conjugated goat antibody. Chemiluminescence reactions were used (WBKLS0100; Millipore) and detected by EZ-Capture MG (AE-9300H-CSP; ATTO).

The nuclei (weight, 50 µg) were incubated on ice for 15 min in a series of buffers: HE (0 mM Mg²⁺ buffer), H10Mg1, and H10Mg5. After incubation, centrifugation was performed to recover nuclei. The nuclear pellets were suspended in a final sample buffer (Laemmli, 1970 (link)) and subjected to 12.5% SDS–PAGE. Subsequently, the gels were subjected to Coomassie brilliant blue (CBB) staining and Western blotting using the following antibodies: mouse anti-Rad21 (05-908; Millipore), rabbit anti-CTCF (07-729; Millipore), rabbit anti-Smc2 (ab10412; Abcam), rabbit anti-H2B (ab1790; Abcam), rabbit anti–acetyl histone H4 (06-866; Millipore), rabbit anti–acetyl histone H3 (06-599; Millipore), and goat anti–lamin A/C (sc-6215; Santa Cruz Biotechnology).