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 25μg of chromatin, 2μg of ab177188 (blue), and 20μl of Anti rabbit IgG sepharose beads. 2μg of rabbit normal IgG was added to the beads control (yellow). The immunoprecipitated DNA was quantified by real time PCR (SYBR approach). Primers are located in the first kb of the transcribed region.

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized HeLa cells labeling Histone H4 (mono methyl K20) with ab177188 at 1/100 dilution, followed by Goat anti-rabbit IgG (Alexa Fluor® 488) (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 (AlexaFluor®594 Goat anti-Mouse secondary) at 1/500 dilution (red).
The negative controls are as follows:
1. ab177188 at 1/100 dilution followed by ab150120 (AlexaFluor®594 Goat anti-Mouse secondary) at 1/500 dilution.
2. ab7291 (anti-Tubulin mouse mAb) at 1/500 dilution followed by ab150077 (Alexa Fluor®488 Goat Anti-Rabbit IgG H&L) at 1/400 dilution.

 

All lanes : Anti-Histone H4 (mono methyl K20) antibody [EPR16999] - ChIP Grade (ab177188) at 1/5000 dilution

Lane 1 : Hela whole cell lysates
Lane 2 : NIH/3T3 whole cell lysates

Lysates/proteins at 10 µg per lane.

Predicted band size: 11 kDa
Observed band size: 11 kDa

Blocking buffer/Diluting buffer: 5% NFDM/TBST

ab177188 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 H4 (mono methyl K20) with ab177188 at 1/100 dilution, followed by prediluted Goat Anti-Rabbit IgG H&L (HRP). Nucleus staining on glandular epithelium of colon tissue is observed. Counter stained with Hematoxylin.

Negative control: PBS instead of primary antibody, secondary antibody 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 H4 (mono methyl K20) with ab177188 at 1/100 dilution, followed by prediluted Goat Anti-Rabbit IgG H&L (HRP). Nucleus staining on glandular epithelium of mouse colon tissue is observed. Counter stained with Hematoxylin.

Negative control: PBS instead of primary antibody, secondary antibody 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 rat kidney tissue labeling Histone H4 (mono methyl K20) with ab177188 at 1/100 dilution, followed by prediluted Goat Anti-Rabbit IgG H&L (HRP). Nucleus staining on rat kidney tissue is observed. Counter stained with Hematoxylin.

Negative control: PBS instead of primary antibody, secondary antibody 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.