ab175046 staining Histone H2B (acetyl K5) in HeLa cells. The cells were incubated with 10mM Sodium butyrate (ab120948) for 6 hours (Treated) or solvent-only for control purposes (Non-treated). Cells were fixed with 100% methanol (5min) and then blocked in 1% BSA/10% normal goat serum/0.3M glycine in 0.1%PBS-Tween for 1h. The cells were then incubated with ab175046 at 0.5µg/ml and ab7291 at 1µg/ml overnight at +4°C, followed by a further incubation at room temperature for 1h with an AlexaFluor®488 Goat anti-Rabbit secondary (ab150077) at 2 μg/ml (shown in green) and AlexaFluor®594 Goat anti-Mouse secondary (ab150120) at 2 μg/ml (shown in pseudo colour red). Nuclear DNA was labelled in blue with DAPI.Negative controls: 1– Rabbit primary and anti-mouse secondary antibody; 2 – Mouse primary antibody and anti-rabbit secondary antibody. Controls 1 and 2 indicate that there is no unspecific reaction between primary and secondary antibodies used.
Chromatin was prepared from HeLa cells according to the Abcam X-ChIP protocol. Cells were fixed with formaldehyde for 10 minutes. The ChIP was performed with 25µg of chromatin, 2µg of ab175046 (blue), and 20µl of Protein A/G sepharose beads. No antibody was added to the beads control (yellow). The immunoprecipitated DNA was quantified by real time PCR (Taqman approach for active and inactive loci, Sybr green approach for heterochromatic loci). Primers and probes are located in the first kb of the transcribed region.
All lanes : Anti-Histone H2B (acetyl K5) antibody - ChIP Grade (ab175046) at 1 µg/mlLane 1 : ATCC HeLa Butyrate (0µM, 6hr) Whole Cell Lysate (ab189243)Lane 2 : ATCC HeLa Butyrate (10µM, 6hr) Whole Cell Lysate (ab189242)Lane 3 : NIH 3T3 (Mouse embryonic fibroblast cell line) Whole Cell LysateLysates/proteins at 2.5 µg per lane.SecondaryGoat Anti-Rabbit IgG H&L (Alexa Fluor® 790) (ab175781) at 1/10000 dilution