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, 5µg of ab32521 (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 H3 (di methyl K9) antibody [Y49] - ChIP Grade (ab32521) at 1/1000 dilution

Lane 1 : HeLa cell lysate
Lane 2 : recombinant Histone H3

Predicted band size: 15 kDa
Observed band size: 17 kDa why is the actual band size different from the predicted?

Immunocytochemistry/Immunofluorescence analysis of HeLa (human cervix adenocarcinoma) labelling Histone H3 (di methyl K9) with purified ab32521 at 1/500. Cells were fixed with 4% PFA and permeabilized with 0.1% Triton X-100. An Alexa Fluor^® 488-conjugated goat anti-rabbit IgG (1/1000) was used as the secondary antibody (Ab150077). Nuclei counterstained with DAPI (blue).

Control: PBS only

Flow Cytometry analysis of HeLa (human cervix adenocarcinoma) cells labeling Histone H3 (di methyl K9) with purified ab32521 at 1/220 dilution (10ug/mL) (red). Cells were fixed with 4% paraformaldehyde and permeabilised with 90% methanol. A Goat anti rabbit IgG (Alexa Fluor^® 488) (1/2000 dilution) was used as the secondary antibody. Rabbit monoclonal IgG (Black) was used as the isotype control, cells without incubation with primary antibody and secondary antibody (Blue) were used as the unlabeled control.

ab32521 (1/500) staining Histone H3 di-methyl K9 in HeLa cells (green). Cells were fixed in paraformaldehyde, permeabilised with 0.5% Triton X100 and counterstained with DAPI in order to highlight the nucleus (red). For further experimental details, please refer to Abreview.

See Abreview