Anti-Histone H3 (di methyl K79) antibody - ChIP Grade (ab3594)
Key features and details
- Rabbit polyclonal to Histone H3 (di methyl K79) - ChIP Grade
- Suitable for: ChIP, WB, ICC/IF, IHC-P, PepArr
- Reacts with: Cow, Human
- Isotype: IgG
Overview
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Product name
Anti-Histone H3 (di methyl K79) antibody - ChIP Grade
See all Histone H3 primary antibodies -
Description
Rabbit polyclonal to Histone H3 (di methyl K79) - ChIP Grade -
Host species
Rabbit -
Specificity
ab3594 detects a 17 kDa band in single lane Western Blot. Peptide inhibition in Western Blot hasn't been processed. Modification specificity is determined by Peptide Array. ab3594 binds strongly to the Histone H3 dii methyl K79. In Peptide Array ab3594 also partially binds to mono methyl K79 and tri methyl K79 peptides. -
Tested Applications & Species
See all applications and species dataApplication Species ChIP HumanICC/IF HumanIHC-P HumanWB Cow
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Immunogen
Synthetic peptide within Human Histone H3 aa 50 to the C-terminus (di methyl K79) conjugated to keyhole limpet haemocyanin. The exact sequence is proprietary.
(Peptide available asab4556) -
General notes
Learn about ChIP assay kits, other ChIP antibodies, protocols and more in the ChIP assay guide.
Images
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Immunocytochemistry/ Immunofluorescence - Anti-Histone H3 (di methyl K79) antibody - ChIP Grade (ab3594) Tao J. et al PLoS One. 2017 Jun 20;12(6):e0179436. doi: 10.1371/journal.pone.0179436. eCollection 2017. Reproduced under the Creative Commons license http://creativecommons.org/licenses/by/4.0/Verification of H3K79me2 antibody specificity.
The commercial H3K79me2 primary antibody was preincubated with (+) or without (−) antigen peptide (Abcam, catalog no. ab4556, v/v = 5:1) at room temperature for 1.5 h before the incubation with IVF porcine blastocysts. H3K79me2 signals were observed in blastocysts using unabsorbed primary antibody. By contrast, H3K79me2 signals were absent in blastocysts using pre-absorbed primary antibody. H3K79me2 antibody was localized with Alexa Flour 488-conjugated secondary antibody (green). DNA was stained with propidium iodide (red). Bottom panels showed the merged images (yellow) between H3K79me2 signals (green) and DNA staining (red). Scale bars = 50μm.
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ChIP - Anti-Histone H3 (di methyl K79) antibody - ChIP Grade (ab3594)Chromatin was prepared from U-2 OS (Human bone osteosarcoma epithelial cell line) 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 ab3594 (blue), and 20 µl of protein A/G sepharose beads. No antibody was added to the beads control (yellow). The immunoprecipitated DNA was quantified by real time PCR (Taqman approach). Primers and probes are located in the first kb of the transcribed region.
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Western blot - Anti-Histone H3 (di methyl K79) antibody - ChIP Grade (ab3594) This image is courtesy of an anonymous AbreviewAnti-Histone H3 (di methyl K79) antibody - ChIP Grade (ab3594) at 20 µg + HeLa cell lysate at 20 µg
Secondary
HRP-conjugated Goat anti-rabbit IgG polyclonal at 1/2000 dilution
Developed using the ECL technique.
Performed under reducing conditions.
Predicted band size: 15 kDa
Observed band size: 15 kDa
Exposure time: 1 minute
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Immunocytochemistry/ Immunofluorescence - Anti-Histone H3 (di methyl K79) antibody - ChIP Grade (ab3594)ICC/IF image of ab3594 stained HeLa (Human epithelial adenocarcinoma cell line) cells. The cells were 100% methanol fixed (5 min) and then incubated in 1% BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1h to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab3594, 1µg/ml) overnight at +4°C. The secondary antibody (green) was Alexa Fluor® 488 goat anti-rabbit IgG (H+L) used at a 1/1000 dilution for 1h. Alexa Fluor® 594 WGA was used to label plasma membranes (red) at a 1/200 dilution for 1h. DAPI was used to stain the cell nuclei (blue) at a concentration of 1.43µM. This antibody also gave a positive result in 100% methanol fixed (5 min) Hek293, HepG2 and MCF7 cells at 1µg/ml, and in 4% PFA fixed (10 min) HeLa, Hek293, HepG2 and MCF7 cells at 1µg/ml.
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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Histone H3 (di methyl K79) antibody - ChIP Grade (ab3594)IHC image of Histone H3 (di methyl K79) staining in human breast carcinoma FFPE section, performed on a BondTM system using the standard protocol F. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6, epitope retrieval solution 1) for 20 mins. The section was then incubated with ab3594, 1µg/ml, for 8 mins at room temperature and detected using an HRP conjugated compact polymer system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX.
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Peptide Array - Anti-Histone H3 (di methyl K79) antibody - ChIP Grade (ab3594)All batches of ab3594 are tested in Peptide Array against peptides to different Histone H3 modifications. Six dilutions of each peptide are printed on to the Peptide Array in triplicate and results are averaged before being plotted on to a graph. Results show strong binding to Histone H3 - di methyl K79 peptide (ab4556), indicating that this antibody specifically recognises the Histone H3 - di methyl K79 modification.
ab4556 - Histone H3 - di methyl K79
ab4555 - Histone H3 - mono methyl K79
ab4557 - Histone H3 - tri methyl K79
ab4560 - Histone H4 - di methyl K79
ab1772 - Histone H3 - di methyl K9
ab4558 - Histone H3 - unmodified
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Western blot - Anti-Histone H3 (di methyl K79) antibody - ChIP Grade (ab3594)Anti-Histone H3 (di methyl K79) antibody - ChIP Grade (ab3594) at 1 µg/ml + Calf Thymus Histone Preparation Nuclear Lysate (ab121) at 0.5 µg
Secondary
Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/10000 dilution
Developed using the ECL technique.
Performed under reducing conditions.
Predicted band size: 15 kDa
Observed band size: 17 kDa why is the actual band size different from the predicted?
Exposure time: 30 seconds
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ChIP - Anti-Histone H3 (di methyl K79) antibody - ChIP Grade (ab3594) This image is courtesy of an anonymous AbreviewChIP analysis using ab3594 binding Histone H3 in HeLa (Human epithelial adenocarcinoma cell line) cell nuclear lysate. Cells were cross-linked for 10 minutes with formaldehyde. Samples were incubated with primary antibody (5µg/µg chromatin) for 12 hours at 4°C. Protein binding was detected using real-time PCR.
Positive control: 5'UTR of transcribed gene.
Negative Control: Intergenic region.
