Chromatin was prepared from HeLa cells according to the Abcam X-ChIP protocol. Cells were fixed with formaldehyde for 10min.The ChIP was performed with 25 µg of chromatin, 5 µg of ab177184 (red), and 20 µl of Protein A/G Sepharose beads. 5 μg of rabbit normal IgG was added to the beads control (grey). The immunoprecipitated DNA was quantified by real time PCR (Taqman approach for active and inactive loci). Primers and probes are located in the first kb of the transcribed region

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized HeLa (Human epithelial cells from cervix adenocarcinoma) cells labeling Histone H3 (di methyl K79) with ab177184 at 1/2000 dilution, followed by Goat anti-rabbit Alexa Fluor® 488 (ab150077) secondary antibody at 1/500 dilution (green). Nuclear staining on HeLa cell line is observed. The nuclear counter stain is DAPI (blue). Tubulin is detected with ab7291 (anti-Tubulin mouse mAb) at 1/1000 dilution and ab150120 (goat anti-mouse AlexaFluor®594 secondary) at 1/500 dilution (red).
The negative controls are as follows;
1. ab177184 at 1/2000 dilution followed by ab150120 (goat anti-mouse AlexaFluor®594 secondary) at 1/500 dilution.
2. ab7291 (anti-Tubulin mouse mAb) at 1/1000 dilution followed by ab150077 (goat anti-rabbit Alexa Fluor®488; IgG H&L) at 1/400 dilution.

All lanes : Anti-Histone H3 (di methyl K79) antibody [EPR17467] - ChIP Grade (ab177184) at 1/2500 dilution

Lane 1 : HeLa (Human epithelial cells from cervix adenocarcinoma) whole cell lysate
Lane 2 : NIH/3T3 (Mouse embyro fibroblast cells) whole cell lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated at 1/1000 dilution

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


Exposure time: 3 minutes

Blocking/Dilution buffer: 5% NFDM/TBST.

ab177184 was tested in Peptide Array against 501 different modified and unmodified histone peptides; each peptide is printed on the array at six concentrations (each in triplicate).
Circle area represents affinity between the antibody and a peptide: all antigen-containing peptides are displayed as red circles, all other peptides as blue circles. The affinity is calculated as area under curve when antibody binding values are plotted against the corresponding peptide concentration. Each circle area is normalized to the peptide with the strongest affinity.
The complete dataset, including full list of all peptides and information on the position of each peptide in the diagram, can be downloaded here.

Immunohistochemical analysis of paraffin-embedded Human colon tissue labeling Histone H3 (di methyl K79) with ab177184 at 1/2000 dilution, followed by Goat Anti-Rabbit HRP (IgG H&L) at 1/500 dilution (ab97051). Nucleus staining on glandular epithelium of Human colon tissue is observed. Counter stained with Hematoxylin.

Negative control: Using PBS instead of primary antibody.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

Immunohistochemical analysis of paraffin-embedded Mouse liver tissue labeling Histone H3 (di methyl K79) with ab177184 at 1/2000 dilution, followed by Goat Anti-Rabbit HRP (IgG HRP) at 1/500 dilution (ab97051). Nucleus staining mouse liver tissue is observed. Counter stained with Hematoxylin.

Negative control: Using PBS instead of primary antibody.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

Immunohistochemical analysis of paraffin-embedded Rat pancreas tissue labeling Histone H3 (di methyl K79) with ab177184 at 1/2000 dilution, followed by Goat Anti-Rabbit HRP (IgG H&L) at 1/500 dilution (ab97051). Nucleus staining on rat pancreas tissue is observed. Counter stained with Hematoxylin.

Negative control: Using PBS instead of primary antibody.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

Chromatin was prepared from Hela (Human epithelial cells from cervix adenocarcinoma) cells according to the Abcam X-ChIP protocol. Cells were fixed with formaldehyde for 10min. The ChIP was performed with 25μg of chromatin, 10μg of ab177184 (blue), and 20μl of Anti rabbit IgG sepharose beads. 10μg of Rabbit normal IgG was added to the beads control (yellow). The immunoprecipitated DNA was quantified by real time PCR (SYBR approach). Primers and probes are located in the first kb of the transcribed region.