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Synthetic peptide corresponding to Human Histone H3 aa 1-100 (tri methyl K4) conjugated to Keyhole Limpet Haemocyanin (KLH) (Cysteine residue).
(Peptide available as
Our Abpromise guarantee covers the use of ab1012 in the following tested applications.
The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
|ChIP||Use 2-5 µg for 25 µg of chromatin.|
|Flow Cyt||Use 1µg for 106 cells.
ab170192 - Mouse monoclonal IgG2b, is suitable for use as an isotype control with this antibody.
|ICC/IF||Use at an assay dependent concentration.|
|IHC-Fr||Use at an assay dependent concentration. PubMed: 20047469|
|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 K4) peptide (ab1342). NOT SUITABLE for blocking with milk. Block in 5% BSA for 1 hour. Our labs have investigated the blocking conditions for this antibody and found that milk significantly decreases the signal and is therefore not a suitable blocking agent for this antibody (see image below).|
|ICC||Use a concentration of 5 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 25 µg of chromatin, 5 µg of ab1012 (blue), and 20 µl 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). Primers and probes are located in the first kb of the transcribed region.
ICC/IF image of ab1012 stained HeLa cells. The cells were 4% PFA fixed (10 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 (ab1012, 5µg/ml) overnight at +4°C. The secondary antibody (green) was Alexa Fluor® 488 goat anti-mouse IgG (H+L) used at a 1/1000 dilution for 1h. Alexa Fluor® 594 WGA was used to label plasma membranes (red) at a 1/200 dilution for 1h. DAPI was used to stain the cell nuclei (blue) at a concentration of 1.43µM. This antibody also gave a positive result in 4% PFA fixed (10 min) HepG2, Hek293 and MCF7 cells at 5µg/ml, and in 100% methanol fixed (5 min) HeLa, Hek293, HepG2 and MCF7 cells at 5µg/ml.
ELISA using ab1012 at varying antibody concentrations.
Curve_SPL4 indicates binding to the tri methyl K4 peptide ab1342. Curve SPL3 indicates partial binding to the di methyl K4 peptide ab7768. There is very weak cross-reactivity with the mono methyl K4 peptide ab1340 (Curve_SPL2).
Binding to the following peptides was not seen: SPL1 unmodified Histone H3, SPL5 Histone H3 mono methyl K9, SPL6 Histone H3 di methyl K9, SPL7 Histone H3 tri methyl K9, SPL8 Histone H3 mono methyl K27, SPL9 Histone H3 di methyl K27, SPL10 Histone H3 tri methyl K27.
Overlay histogram showing HeLa cells stained with ab1012 (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 (ab1012, 1µg/1x106 cells) for 30 min at 22ºC. The secondary antibody used was DyLight® 488 goat anti-mouse IgG (H+L) (ab96879) at 1/500 dilution for 30 min at 22ºC. Isotype control antibody (black line) was mouse IgG2b [PLPV219] (ab91366, 2µg/1x106 cells) used under the same conditions. Acquisition of >5,000 events was performed.
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