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Synthetic peptide within Human Histone H3 aa 1-100 (tri methyl K36) conjugated to Keyhole Limpet Haemocyanin (KLH). The exact sequence is proprietary.
(Peptide available as
For detection of methylated histone H3.
Our Abpromise guarantee covers the use of ab9050 in the following tested applications.
The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
|ICC/IF||Use a concentration of 0.1 - 1 µg/ml.|
|CHIPseq||Use at an assay dependent concentration. PubMed: 19581485|
|WB||Use a concentration of 1 µg/ml. Detects a band of approximately 15 kDa (predicted molecular weight: 15 kDa).|
|ChIP||Use 4µg for 106 cells.|
|ChIP/Chip||Use at an assay dependent concentration.|
|IHC-P||Use a concentration of 0.5 - 10 µg/ml.|
Chromatin was prepared from U2OS 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 ab9050 (blue), and 20µl of Protein A/G sepharose beads. No antibody was added to the beads control (yellow). The immunoprecipitated DNA was quantified on the GAPDH (active) and MYO-D (inactive) promoters and over the ý-Actin gene (active). Schematic diagram of the ý-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.
ab9050 stained in Hela cells. Cells were fixed with 100% methanol (5min) at room temperature and incubated with PBS containing 10% goat serum, 0.3 M glycine, 1% BSA and 0.1% triton for 1h at room temperature to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody ab9050 at 0.1µg/ml and ab7291 (Mouse monoclonal [DM1A] to alpha Tubulin - Loading Control) at 1/1000 dilution overnight at +4°C. The secondary antibodies were ab150120 (pseudo-colored red) and ab150081 (colored green) used at 1 ug/ml for 1hour at room temperature. DAPI was used to stain the cell nuclei (colored blue) at a concentration of 1.43µM for 1hour at room temperature.
ab9050 staining Histone H3 (tri-methyl K36) in HeLa cells. The cells were fixed with 100% methanol (5min) and then blocked in 1% BSA/10% normal goat serum/0.3M glycine in 0.1%PBS-Tween for 1h. The cells were then incubated with ab9050 at 0.1µg/ml and ab7291 (anti beta-Tubulin) at 1µg/ml overnight at +4°C, followed by a further incubation at room temperature for 1h with a goat anti -rabbit AlexaFluor®488 secondary (ab150081) at 2 μg/ml (shown in green) and a goat anti-mouse AlexaFluor®594 (ab150120) at 2 μg/ml (shown in pseudo color red). Nuclear DNA was labelled in blue with DAPI.
Negative controls: 1– Rabbit primary antibody and anti-mouse secondary antibody; 2 – Mouse primary antibody and anti-rabbit secondary antibody. Controls 1 and 2 indicate that there is no unspecific reaction between primary and secondary antibodies used.
ab9050 staining mouse kidney tissue sections by IHC-P. The section was formaldehyde fixed and subjected to heat mediated antigen retrieval in pH 6.0 citrate buffer prior to being blocked with 5% serum for 30 minutes at 20°C. The primary antibody was diluted 1/500 and incubated for 45 minutes at 20°C. A HRP conjugated goat anti-rabbit was used as the secondary
ab9050 staining Histone H3 (tri methyl K36) in Human HEK293 cells by ICC/IF (Immunocytochemistry/immunofluorescence). Cells were fixed with paraformaldehyde, permeabilized with Triton X-100 and blocked with 1% serum for 10 minutes at 21°C. Samples were incubated with primary antibody (1/500) for 10 hours at 21°C. A FITC-conjugated goat anti-rabbit IgG polyclonal (1/500) was used as the secondary antibody.
ab9050 staining rat liver tissue sections by IHC-P. The section was formaldehyde fixed and subjected to heat mediated antigen retrieval in pH 6.0 citrate buffer prior to being blocked with 5% serum for 30 minutes at 45°C. The primary antibody was diluted 1/500 and incubated for 45 minutes at 20°C. A HRP conjugated goat anti-rabbit was used as the secondary