Chromatin was prepared from U-2 OS (Human bone osteosarcoma epithelial cell line) cells according to the Abcam X-ChIP protocol. Cells were fixed with formaldehyde for 10 min. The  ChIP was performed with 25 µg of chromatin, 2 µg of  ab3594 (blue), and 20 µl 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). Primers and probes are located in the first kb of the transcribed region.

Anti-Histone H3 (di methyl K79) antibody - ChIP Grade (ab3594) at 20 µg + HeLa cell lysate at 20 µg

Secondary
HRP-conjugated Goat anti-rabbit IgG polyclonal at 1/2000 dilution

Developed using the ECL technique.

Performed under reducing conditions.

Predicted band size: 15 kDa
Observed band size: 15 kDa


Exposure time: 1 minute

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ICC/IF image of ab3594 stained HeLa (Human epithelial adenocarcinoma cell line) cells. The cells were 100% methanol fixed (5 min) and then incubated in 1% BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1h to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab3594, 1µg/ml) overnight at +4°C. The secondary antibody (green) was Alexa Fluor® 488 goat anti-rabbit IgG (H+L) used at a 1/1000 dilution for 1h. Alexa Fluor® 594 WGA was used to label plasma membranes (red) at a 1/200 dilution for 1h. DAPI was used to stain the cell nuclei (blue) at a concentration of 1.43µM. This antibody also gave a positive result in 100% methanol fixed (5 min) Hek293, HepG2 and MCF7 cells at 1µg/ml, and in 4% PFA fixed (10 min) HeLa, Hek293, HepG2 and MCF7 cells at 1µg/ml.

IHC image of Histone H3 (di methyl K79) staining in human breast carcinoma FFPE section, performed on a Bond^TM system using the standard protocol F. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6, epitope retrieval solution 1) for 20 mins. The section was then incubated with ab3594, 1µg/ml, for 8 mins at room temperature and detected using an HRP conjugated compact polymer system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX.

All batches of ab3594 are tested in Peptide Array against peptides to different Histone H3 modifications. Six dilutions of each peptide are printed on to the Peptide Array in triplicate and results are averaged before being plotted on to a graph. Results show strong binding to Histone H3 - di methyl K79 peptide (ab4556), indicating that this antibody specifically recognises the Histone H3 - di methyl K79 modification.

ab4556 - Histone H3 - di methyl K79

ab4555 - Histone H3 - mono methyl K79

ab4557 - Histone H3 - tri methyl K79

ab4560 - Histone H4 - di methyl K79

ab1772 - Histone H3 - di methyl K9

ab4558 - Histone H3 - unmodified

Anti-Histone H3 (di methyl K79) antibody - ChIP Grade (ab3594) at 1 µg/ml + Calf Thymus Histone Preparation Nuclear Lysate (ab121) at 0.5 µg

Secondary
Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/10000 dilution

Developed using the ECL technique.

Performed under reducing conditions.

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


Exposure time: 30 seconds

ChIP analysis using ab3594 binding Histone H3 in HeLa (Human epithelial adenocarcinoma cell line) cell nuclear lysate. Cells were cross-linked for 10 minutes with formaldehyde. Samples were incubated with primary antibody (5µg/µg chromatin) for 12 hours at 4°C. Protein binding was detected using real-time PCR.
Positive control: 5'UTR of transcribed gene.
Negative Control: Intergenic region.

See Abreview