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Synthetic peptide conjugated to KLH derived from within residues 1 - 100 of Human Histone H3, mono methylated at K4.
(Peptide available as ab1340.)
Our Abpromise guarantee covers the use of ab8895 in the following tested applications.
The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
|IP||Use at an assay dependent concentration.|
|Flow Cyt||1/100. ab171870-Rabbit polyclonal IgG, is suitable for use as an isotype control with this antibody.|
|Electron Microscopy||Use at an assay dependent concentration. PubMed: 20543957|
|ICC/IF||Use at an assay dependent concentration.|
|IF||Use a concentration of 1 µg/ml.
Works better if cells are fixed with methanol.
|ICC||Use at an assay dependent concentration.|
|ChIP||Use 2 µg for 25 µg of chromatin.|
|WB||1/500. Detects a band of approximately 17 kDa (predicted molecular weight: 15 kDa).Can be blocked with Human Histone H3 (mono methyl K4) peptide (ab1340).|
|CHIPseq||Use at an assay dependent concentration. PubMed: 22196736|
|IHC-P||Use a concentration of 0.5 µg/ml. Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.|
|ChIP/Chip||Use at an assay dependent concentration.|
ab8895 staining Histone H3 (mono methyl K4) 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 ab8895 at 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 secondary (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.
IHC image of ab8895 staining Histone H3 (mono methyl K4) in human colon formalin fixed paraffin embedded tissue sections, performed on a Leica Bond. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6, epitope retrieval solution 1) for 20 mins. The section was then incubated with ab8895, 0.5µg/ml, for 15 mins at room temperature and detected using an HRP conjugated compact polymer system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX. No primary antibody was used in the negative control (shown on the inset).
For other IHC staining systems (automated and non-automated) customers should optimize variable parameters such as antigen retrieval conditions, primary antibody concentration and antibody incubation times.
ab8895 is specific for mono-methylated Lysine 4 of histone H3 and does not recognize di- or tri-methyl Lysine 4 nor methylation at Lysine 9. This is shown in lane 2 where the activity of the antibody is specifically blocked by the addition of the immunizing peptide (ab1340).
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 ab8895 (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 and ALDOA (active) and MYO-D (inactive) promoters and over the y-Actin gene (active). Schematic diagram of the y-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.
Indian muntjac fibroblast cells - interphase (top left) and prophase (top right and below), stained with:
Mono Methyl K4 antibody, ab8895, (green)
DAPI: red, top left and right; blue, below
Phospho Ser 10 antibody: red (below) and blue (top right)
The perinuclear and perinucleolar heterochromatin domains do not contain Mono Methyl K4. The Mono Methyl K4, rather, is distributed as small nuclear foci primarily found between DAPI-intense regions of the nucleus.
ab8895 staining mouse embryonic stem cells by flow cytometry. The ES cell colonies were trypsinized and the cells permeabilized before being stained with ab8895 (0.25ug/1.5 x 105 cells). A PE conjugated goat anti-rabbit antibody was used as the secondary.
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