Chromatin was prepared from HeLa (human epithelial cell line from cervix adenocarcinoma) 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 ab176877 (red), or 2 µg of rabbit normal IgG ab172730 (gray) and 20 µl of Protein A/G sepharose beads. The immunoprecipitated DNA was quantified by real time PCR (Taqman approach for active and inactive loci, Sybr green approach for heterochromatic loci).

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized HeLa (Human epithelial cell line from cervix adenocarcinoma) cells labeling Histone H3 (mono methyl K4) with ab176877 at 1/1000 dilution, followed by goat anti-rabbit Alexa Fluor^® 488 (IgG) (ab150077) secondary antibody at 1/400 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 1/500 dilution and ab150120 (Alexa Fluor^® 594 goat anti-mouse secondary) at 1/500 dilution (red).
The negative controls are as follows;
1. ab176877 at 1/1000 dilution followed by ab150120 (goat anti-mouse Alexa Fluor^® 594 secondary) at 1/500 dilution.
2. ab7291 (anti-Tubulin mouse mAb) at 1/500 dilution followed by ab150077 (goat anti-rabbit Alexa Fluor^® 488; IgG H&L) at 1/400 dilution.

 

Immunohistochemical analysis of paraffin-embedded rat colon tissue labeling Histone H3 (mono methyl K4) with ab176877 at 1/1000 dilution, followed by prediluted Goat Anti-Rabbit IgG H&L (HRP). Nucleus staining on glandular epithelium of colon tissue is observed. Counterstained with hematoxylin.

Negative control: PBS instead of primary ab, secondary ab is prediluted Goat Anti-Rabbit IgG H&L (HRP).

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

All lanes : Anti-Histone H3 (mono methyl K4) antibody [ERP16597] - ChIP Grade (ab176877) at 1/5000 dilution

Lane 1 : HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell lysates
Lane 2 : NIH/3T3 (Mouse embryo fibroblast cell line) whole cell lysates

Lysates/proteins at 10 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

Blocking/Dilution buffer: 5% NFDM/TBST.

ab176877 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 (mono methyl K4) with ab176877 at 1/1000 dilution, followed by prediluted Goat Anti-Rabbit IgG H&L (HRP).  Nucleus staining on glandular epithelium of colon tissue is observed. Counterstained with hematoxylin.

Negative control: PBS instead of primary ab, secondary ab is prediluted Goat Anti-Rabbit IgG H&L (HRP).

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

Immunohistochemical analysis of paraffin-embedded mouse colon tissue labeling Histone H3 (mono methyl K4) with ab176877 at 1/1000 dilution, followed by prediluted Goat Anti-Rabbit IgG H&L (HRP). Nucleus staining on glandular epithelium of colon tissue is observed. Counterstained with hematoxylin.

Negative control: PBS instead of primary ab, secondary ab is prediluted Goat Anti-Rabbit IgG H&L (HRP).

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