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Synthetic peptide derived from within residues 1 - 100 Histone H3.
(Peptide available as ab1771.)
Our Abpromise guarantee covers the use of ab8896 in the following tested applications.
The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
|WB||Use a concentration of 0.5 - 2 µg/ml. Detects a band of approximately 17 kDa (predicted molecular weight: 15 kDa).Can be blocked with Human Histone H3 (mono methyl K9) peptide (ab1771).|
|ChIP||Use 2-25 µg for µg of chromatin.|
|ICC/IF||Use at an assay dependent concentration.|
|CHIPseq||Use at an assay dependent concentration. PubMed: 22196736|
ab8896 specifically recognises mono-methyl K9 (lane 1) in calf thymus lysate at 17kDa. ab8896 is successfully blocked using the immunizing peptide (lane 2 ab1771), but not the di-methyl K9 (lane 3 ab1772), the tri-methyl K9 (lane 4 ab1773), the mono-methyl K27 (lane 5 ab1780), the di-methyl K27 (lane 6 ab1781), the tri-methyl K27 (lane 7 ab1782), the mono-methyl K4 (lane 8 ab1340) nor the corresponding unmodified peptides (lane 9 ab7228, lane 10 ab2623). This implies that ab8896 is specific to mono-methyl K9.
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 ab8896 (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.
ab8896 staining Histone H3 (mono methyl K9) 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).
ICC/IF image of ab8896 stained HeLa 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 ab8896 at 1µg/ml overnight at +4°C. The secondary antibody (pseudo-colored green) was a goat anti-rabbit Alexa Fluor® 488 (ab150081) IgG (H+L) preadsorbed, used at a 1/1000 dilution for 1h. Alexa Fluor® 594 WGA was used to label plasma membranes (pseudo-colored red) at a 1/200 dilution for 1h at room temperature. DAPI was used to stain the cell nuclei (pseudo-colored blue) at a concentration of 1.43µM for 1hour at room temperature.
The image is a courtesy of an anonymous abreview.
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