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Does not react withCarp
Synthetic peptide within Human Histone H3 aa 1-100 (tri methyl K27) conjugated to Keyhole Limpet Haemocyanin (KLH) (Sulfosuccinimidyl 4-N-maleimidomethyl-cyclohexane-1-carboxylate (Sulfo-SMCC)). The exact sequence is proprietary.
(Peptide available as
Hybridomas were prepared and the resulting clones were positively screened by ELISA against the immunising peptide. Clones were negatively screened against both the corresponding unmodified peptide and also against a peptide corresponding to tri methylated K9 of Histone H3.
Our Abpromise guarantee covers the use of ab6147 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 1 - 5 µg/ml.|
|IHC-P||Use at an assay dependent concentration.|
|ELISA||Use a concentration of 1 µg/ml. PubMed: 17641388|
|WB||Use a concentration of 1 - 5 µg/ml. Detects a band of approximately 17 kDa (predicted molecular weight: 15 kDa).Can be blocked with Human Histone H3 (tri methyl K27) peptide (ab1782).|
|Flow Cyt||Use 1-2µg for 106 cells. Ab170190-Mouse monoclonal IgG1, is suitable for use as an isotype control with this antibody.|
ICC/IF image of ab6147 stained mouse 2 cell embryo. Cells were fixed with formaldehyde, permeabilized with 0.5% Triton, and incubated with ab6147 at 1/50 for 2 hours at 37°C. Blocking was performed in 5% FCS serum for 30 minutes at 37°C. The secondary antibody (green) was Alexa Fluor® 488 goat anti-mouse IgG used at a 1/250 dilution. PI was used to stain the DNA (Red).
ELISA using ab6147.
The red line indicates binding to the tri methyl K27 peptide (ab1782). Weak binding (brown line) was seen to the di methyl K27 peptide (ab1781). Binding to the following peptides was not seen:
Unmodified K27 (ab2623),
mono methyl K27 (ab1780),
mono methyl K9 (ab1771),
di methyl K9 (ab1772),
tri methyl K9 (ab1773),
mono methyl K4 (ab1340),
di methyl K4 (ab7768),
tri methyl K4 (ab1342).
This indicates the specificity of ab6147 for tri methyl K27 of Histone H3.
Overlay histogram showing HeLa cells stained with ab6147 (red line). The cells were fixed with 80% methanol (5 min) and then permeabilized with 0.1% PBS-Tween for 20 min. The cells were then incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions followed by the antibody (ab6147, 1µg/1x106 cells) for 30 min at 22ºC. The secondary antibody used was a goat anti-mouse DyLight® 488 (IgG; H+L) (ab96879) at 1/500 dilution for 30 min at 22ºC. Isotype control antibody (black line) was mouse IgG1 [ICIGG1] (ab91353, 2µg/1x106 cells) used under the same conditions. Acquisition of >5,000 events was performed. This antibody gave a positive signal in HeLa cells fixed with 4% paraformaldehyde (10 min)/permeabilized with 0.1% PBS-Tween for 20 min used under the same conditions.
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