Recombinant Anti-Histone H3 (tri methyl K27) antibody [EPR18607] - BSA and Azide free (ab222481)

This ChIP data was generated using the same anti-Histone H3K27me3 antibody clone, EPR18607, in a different buffer formulation (cat# ab192985).

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 minutes. The ChIP was performed with 25µg of chromatin, 2µg of ab192985 (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 green approach).

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 ab 222481 [EPR18607]. ChIP DNA was sequenced on the Illumina NextSeq 500 to a depth of 10 million sequence tags. The image shows binding across a region of chromosome 1 (RLF gene). ChIP-Seq validation performed by Active Motif, Carlsbad, CA

Peptide array analysis was performed using ab192985 at a concentration of 0.1ug/ml, followed by Goat Anti-Rabbit IgG, (H+L), Fluo 647nm conjugated secondary antibody at a 1/50000 dilution.

ab192985 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.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab192985).

ELISA analysis was performed on 1ug/ml of antigen using ab192985 at a concentration range of 0-0.25ug/ml, followed by Alkaline Phosphatase-conjugates AffiniPure Goat anti-rabbit IgG (H&L) secondary antibody at a 1/2500 dilution.

All batches of ab192985 are tested in ELISA against peptides to different Histone H3 modifications. Results show strong binding to Histone H3 - tri methyl K27 immunizing peptide, indicating that this antibody specifically recognizes the Histone H3 - tri methyl K27 modification. Weak binding (14%) was also detected against H3 - di methyl K27 modification.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab192985).

Immunohistochemical analysis of paraffin-embedded human colon tissue labeling Histone H3 (tri methyl K27) with ab192985 at 1/500 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution. Nuclear staining on human colon tissue is observed. Counter stained with Hematoxylin.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab192985).

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 (tri methyl K27) with ab192985 at 1/500 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution. Nuclear staining on mouse colon tissue is observed. Counter stained with Hematoxylin.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab192985).

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 H3 (tri methyl K27) with ab192985 at 1/500 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution. Nuclear staining on rat kidney tissue is observed. Counter stained with Hematoxylin.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab192985).

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

This ICC/IF data was generated using the same anti-Histone H3K27me3 antibody clone, EPR18607, in a different buffer formulation (cat# ab192985).

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized HeLa (Human epithelial cells from cervix adenocarcinoma) cells labeling Histone H3 (tri methyl K27) with ab192985 at 1/1000 dilution, followed by Goat Anti-Rabbit IgG (Alexa Fluor® 488) (ab150077) secondary antibody at 1/1000 dilution (green). Confocal image showing nuclear staining on HeLa cell line. The nuclear counterstain is DAPI (blue).

Tubulin is detected with Anti-alpha Tubulin mouse MAb (ab7291) at 1/1000 dilution, followed by Goat Anti-Mouse IgG H&L (Alexa Fluor^® 594) (ab150120) secondary antibody at 1/1000 dilution (red).

The negative controls are as follows:-
-ve control 1: ab192985 at 1/1000 dilution, followed by Goat Anti-Mouse IgG H&L (Alexa Fluor^® 594) (ab150120) secondary antibody at 1/1000 dilution.
-ve control 2: Anti-alpha Tubulin mouse MAb (ab7291) at 1/1000 dilution, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution.