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Synthetic peptide conjugated to KLH derived from within residues 1 - 100 of Human Histone H3, di methylated at K27.
(Peptide available as ab1781.)
Our Abpromise guarantee covers the use of ab24684 in the following tested applications.
The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
|Dot Blot||Use at an assay dependent concentration.|
|WB||Use a concentration of 0.5 µg/ml. Detects a band of approximately 18 kDa (predicted molecular weight: 15.2 kDa).Can be blocked with Human Histone H3 (di methyl K27) peptide (ab1781).|
|IP||Use at 80 µg/mg of lysate.|
|IHC-P||Use a concentration of 1 µg/ml.|
|IHC - Wholemount||Use at an assay dependent concentration.|
|ICC/IF||Use a concentration of 1 µg/ml.|
|ChIP||Use 2-25 µg for µg of chromatin.|
The peptide competition assay shows that this antibody is very specific for Histone H3 di-methylated at K27. It does not recognise Histone H3 mono
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 ab24684 (blue), and 20µl 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 for active and inactive loci, Sybr green approach for heterochromatic loci). Primers and probes are located in the first kb of the transcribed region.
ab24684 staining Histone H3 (di methyl K27) 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 (1/600) for 12 hours at 4°C. An Alexa Fluor® 488-conjugated goat polyclonal anti-rabbit IgG (1/1000) was used as the secondary antibody.
Gating Strategy: Isotype negative control (white).
IHC image of Histone H3 (di methyl K27) staining in Human normal skin formalin fixed paraffin embedded tissue section, performed on a Leica 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 ab24684, 1µg/ml, for 15 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.
For other IHC staining systems (automated and non-automated) customers should optimize variable parameters such as antigen retrieval conditions, primary antibody concentration and antibody incubation times.
Please note: All products are "FOR RESEARCH USE ONLY AND ARE NOT INTENDED FOR DIAGNOSTIC OR THERAPEUTIC USE"