Recombinant Anti-Histone H3 (tri methyl K4) antibody [EPR20551-225] - ChIP Grade (ab213224)

CUT&RUN was performed using the ChIC/CUT&RUN pAG-MNAse ab285373, 10⁵ HeLa cells and 2µg of ab213224 [EPR20551-225]. The resulting DNA was sequenced on the NovaSeq 6000 (PE150).

Additional screenshots of mapped reads can be downloaded here.

CUT&Tag-seq was performed using 200,000 Oli-neu (Oligodendrocyte progenitor) cells. Cells were permeabilized with 0.05% Digitonin and 0.01% NP-40 for 3 minutes. A 1:100 dilution of Anti-Histone H3 (tri methyl K4) antibody [EPR20551-225] - ChIP Grade was used, along with a Guinea pig anti-rabbit Secondary. DNA was seg using Illumina NovaSeq S Prime to a depth of 22 million reads.

Positive control Sox10 chr15:79,091,798-79,329,947 (Mouse mm10 genome used).

This image is courtesy of Dr Marek Bartosovic, Gonçalo Castelo-Branco Group, Karolinska Institutet.

Chromatin was prepared from HeLa cells. Cells were fixed with 1% formaldehyde for 10 minutes. ChIP was performed with 10⁷ cells and 4 µg of Anti-Histone H3 (tri methyl K4) antibody [EPR20551-225] - ChIP Grade. ChIP DNA was sequenced on the Illumina NovaSeq 6000 to a depth of 30 million reads.

Additional screenshots of mapped reads can be downloaded here.

Chromatin was prepared from Hela (human epithelial cel 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 ab213224(red), and 20 μl of Anti-rabbit IgG sepharose beads. 2 μg of Rabbit normal IgG was added to the beads control (grey). The immunoprecipitated DNA was quantified by real time PCR (Taqman approach). Primers and probes are located in the first kb of the transcribed region.

Chromatin was prepared from HeLa cells. Cells were fixed with 1% formaldehyde for 10 minutes. ChIP was performed with 30 µg of chromatin and 4 µg of Anti-Histone H3 (tri methyl K4) antibody [EPR20551-225] - ChIP Grade. ChIP DNA was sequenced on the Illumina NextSeq 500 to a depth of 30 million reads. ChIP-Seq validation performed by Active Motif, Carlsbad, CA.

Additional screenshots of mapped reads can be downloaded here.

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1%, Triton X-100-permeabilized NIH/3T3 (mouse embryo fibroblast cell line) cells labeling Histone H3 (tri methyl K4) with ab213224 at 1/500 dilution, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution (green). Positive nuclear staining on NIH/3T3 cell lines.

The nuclear counter stain is DAPI (blue). Tubulin is detected with Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594) (ab195889) (red) at 1/200 dilution.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution.

Blocking/Dilution buffer: 5% BSA/TBST.

We recommend to use 2% BSA as blocking and antibody dilution buffer.

ab213224 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.

Histone H3 (tri methyl K4) was immunoprecipitated from 0.35 mg of NIH/3T3 (mouse embryo fibroblast cell line) lysate with ab213224 at 1/30 dilution. Western blot was performed from the immunoprecipitate using ab213224 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP) (ab131366), was used for detection at 1/5000 dilution

Lane 1: NIH/3T3 whole cell lysate 10 µg (Input). 

Lane 2: ab213224 IP in NIH/3T3 whole cell lysate. 

Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab213224 in NIH/3T3 whole cell lysate.

Exposure time: 3 minutes.

Blocking and dilution buffer and concentration: 5% BSA/TBST.

Dot blot analysis of Histone H3 (tri methyl K4) labeled with ab213224 at 1/1000 dilution.

Lane 1: Histone H3K4Me3 peptide.

Lane 2: Histone H3 unmodified peptide.

Lane 3: Histone H3K(18+K36)Me3 peptide.

Lane 4: Histone H3K18Me3 peptide.

Lane 5: Histone H3K4Me2 peptide.

Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution was used as secondary antibody.

Exposure time: 3 minutes.

Blocking and dilution buffer: 5% NFDM/TBST.

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100-permeabilized HeLa (human epithelial cell line from cervix adenocarcinoma) cells labeling Histone H3 (tri methyl K4) with ab213224 at 1/500 dilution, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution (green). Positive nuclear staining on HeLa cell line. 

The nuclear counter stain is DAPI (blue). Tubulin is detected with Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594) (ab195889) (red) at 1/200 dilution.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution.

Intracellular flow cytometric analysis of 4% paraformaldehyde-fixed, 90% methanol-permeabilized HeLa (humanepithelialcell line from cervix adenocarcinoma) cell line labeling Histone H3 (tri methyl K4) with ab213224at 1/500 dilution (red) compared with a Rabbit IgG, monoclonal [EPR25A] - Isotype Control (ab172730) (black) and an unlabeled control (cells without incubation with primary antibody and secondary antibody) (blue). Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) at 1/2000 dilution was used as the secondary antibody.