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Synthetic peptide conjugated to KLH derived from within residues 50 to the C-terminus of Human Histone H3, di methylated at K79.
(Peptide available as ab4556.)
Our Abpromise guarantee covers the use of ab3594 in the following tested applications.
The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
|ChIP||Use at an assay dependent concentration.|
|ChIP/Chip||Use at an assay dependent concentration.|
|ICC/IF||Use a concentration of 1 µg/ml.|
|WB||Use a concentration of 1 µg/ml. Detects a band of approximately 17 kDa (predicted molecular weight: 15 kDa).Can be blocked with Human Histone H3 (di methyl K79) peptide (ab4556).|
|CHIPseq||Use at an assay dependent concentration. PubMed: 22196736|
|IHC-P||Use a concentration of 1 µg/ml. Perform heat mediated antigen retrieval before commencing with IHC staining protocol.|
|PepArr||Use a concentration of 0.2 - 0.02 µg/ml.|
ab3594 staining Histone H3 (di methyl K79) in human differentiated haematopoietic stem cells by Flow Cytometry. Cells were fixed with paraformaldehyde and permeabilized with permeabilization buffer. The sample was incubated with the primary antibody (1/800) for 12 hours at 4°C. An Alexa Fluor® 488-conjugated goat polyclonal anti-rabbit IgG (1/1500) was used as the secondary antibody.
Gating Strategy: Isotype negative control (white).
This image is courtesy of an anonymous Abreview
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
ChIP analysis using ab3594 binding Histone H3 in HeLa 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.
Chromatin was prepared from U2OS 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.
Please note: All products are "FOR RESEARCH USE ONLY AND ARE NOT INTENDED FOR DIAGNOSTIC OR THERAPEUTIC USE"