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Synthetic peptide conjugated to KLH derived from within residues 1 - 100 of Human Histone H3.
(Peptide available as ab1784.)
Our Abpromise guarantee covers the use of ab9049 in the following tested applications.
The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
|IHC-P||Use a concentration of 1 µg/ml. Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.|
|Flow Cyt||Use at an assay dependent concentration.|
|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 K36) peptide (ab1784).|
|ChIP||Use 2-5 µg for 25 µg of chromatin.|
IHC image of ab9049 staining Histone H3 (di methyl K36) in human colon formalin fixed paraffin embedded tissue sections, performed on a Leica Bond. 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 ab9049, 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. No primary antibody was used in the negative control (shown on the inset).
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.
ab9049 staining Histon H3 (di methyl K36) in Mouse Neuro-2a neuroblastoma cultured cells by ICC/IF (Immunocytochemistry/immunofluorescence). Cells were fixed with formaldehyde, permeabilized with 0.3% Triton X-100 and blocked with 1% BSA for 1 hour at 25°C. Samples were incubated with primary antibody (1/100) for 16 hours at 4°C. A FITC-conjugated Goat anti-rabbit IgG polyclonal (1/1000) was used as the secondary antibody.
Chromatin was prepared from Hela 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 ab9049(blue), and 20 µl of Protein A/G sepharose beads. No antibody was added to the beads control (yellow). The immunoprecipitated DNA was quantified on the GAPDH and RPL30 (active) promoters and over the g-Actin gene (active). Schematic diagram of the g-Actin gene is shown on the top of the figure. Black boxes represent exons and thin lines represent introns. PCR products are depicted as bars under the gene.
Image courtesy of an anonymous Abreview.
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