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For detection of Histone H3 specifically methylated at position Lys 4. This antibody was used in a screen by Dover et al, (2002) to isolate yeast mutants that are unable to methylate Lysine 4. In immunofluorescence, this antibody detects foci in the nucleus that are non-colocalising with condensed chromatin. The perinuclear and perinucleolar heterochromatin are not stained with this antibody.
Our Abpromise guarantee covers the use of ab7766 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 µg for 25 µg of chromatin.|
|Dot blot||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 K4) peptide (ab7768).|
|ChIP/Chip||Use at an assay dependent concentration.|
|PepArr||Use a concentration of 0.2 - 2 µg/ml.|
Chromatin was prepared from HeLa cells according to the Abcam X-ChIP protocol. Cells were fixed with formaldehyde for 10 minutes. The ChIP was performed with 25µg of chromatin, 2µg of ab7766 (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.
ab7766 at 1/200 staining human U2OS (osteosarcoma) cells by ICC/IF. The cells were paraformaldehyde fixed and then stained with the antibody for 1 hour. A Cy2 ® conjugated donkey anti-rabbit antibody was used as the secondary (green). The image shows uniformal staining of the whole nucleus, with several specles found. The insert shows H3-di methyl K4 of tw2 cells only. DAPI nuclear staining is shown in blue.
All batches of ab7766 are tested in Peptide Array against peptides to different Histone H3 modifications. Six dilutions of each peptide are printed on to the Peptide Array in triplicate and results are averaged before being plotted on to a graph. Results show strong binding to Histone H3 - di methyl K4 peptide (ab7768), indicating that this antibody specifically recognises the Histone H3 - di methyl K4 modification.
MCF7 cells were incubated at 37°C for 24h with vehicle control (0 µM) and different concentrations of tranylcypromine hydrochloride (ab120606). Increased expression of Histone 3 K4 di-methyl (ab7766) in MCF7 cells correlates with an increase in tranylcypromine hydrochloride concentration, as described in literature.
Nuclear extracts were prepared with RIPA buffer (containing protease inhibitors and sodium orthovanadate), 10µg of each were loaded on the gel and the WB was run under reducing conditions. After transfer the membrane was blocked for an hour using 5% BSA before being incubated with ab7766 at 1 µg /ml and ab1791 at 1 µg /ml overnight at 4°C. Antibody binding was detected using an anti-rabbit HRP secondary antibody (ab97051) at 1/10000 dilution and visualised using ECL development solution.
ICC/IF image of ab7766 stained HeLa cells. The cells were 4% formaldehyde 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 (ab7766, 0.1µg/ml) overnight at +4°C. The secondary antibody (green) was ab96899, a goat anti-rabbit DyLight® 488 (IgG; H+L) used at a 1/250 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% formaldehyde fixed (10 min) Hek293, HepG2 and MCF7 cells at 0.1µg/ml, and in 100% methanol fixed (5 min) HeLa, Hek293, HepG2 and MCF7 cells at 0.1µg/ml.