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Synthetic peptide corresponding to Human Histone H3 aa 1-100 (tri methyl K4) conjugated to Keyhole Limpet Haemocyanin (KLH).
(Peptide available as
In immunofluorescence, a distinct property of tri methyl lysine 4 is its apparent 'ringing' of regions that appear as nucleoplasmic 'holes'. These represent the positions of splicing factor compartments, which often are easy to identify using only DNA stains in Indian muntjac fibroblasts. These splicing factor compartments are known to be preferentially associated with active genes and highly acetylated histone H3. The antibody, as expected, fails to stain heterochromatin (work by Kirk McManus, lab of Michael Hendzel). The immunofluorescence results suggest this antibody is an exceptional euchromatin probe.
Our Abpromise guarantee covers the use of ab8580 in the following tested applications.
The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
|ChIP/Chip||Use at an assay dependent concentration.|
|PepArr||Use a concentration of 0.2 - 2 µg/ml.|
|ICC||Use at an assay dependent concentration.|
|ChIP||Use 2 µg for 25 µg of chromatin.|
|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 (tri methyl K4) peptide (ab92374).|
|IF||Use a concentration of 1 µg/ml.|
|IP||Use at an assay dependent concentration.|
|CHIPseq||Use at an assay dependent concentration. PubMed: 20952408|
|Flow Cyt||Use at an assay dependent concentration.|
|ICC/IF||1/100 - 1/5000.|
|IHC-P||Use at an assay dependent concentration. PubMed: 17634443|
IHC image of ab8580 staining Histone H3 (tri methyl K4) in normal 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 ab8580, 1/500 dilution, 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.
*Tissue obtained from the Human Research Tissue Bank, supported by the NIHR Cambridge Biomedical Research Centre
ab8580 staining Histone H3 (tri methyl K4) in HeLa cells. All 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 ab4729 at 1/1000 and ab7291 at 1µg/ml overnight at +4°C, followed by a further incubation at room temperature for 1h with an AlexaFluor®488 Goat anti-Rabbit secondary (ab150077) at 2 μg/ml (shown in green) and AlexaFluor®594 Goat anti-Mouse secondary (ab150120) at 2 μg/ml (shown in pseudo color red). Nuclear DNA was labelled in blue with DAPI.
Negative controls: 1– Rabbit primary 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.
Chromatin was prepared from U2OS cells according to the Abcam X-ChIP protocol. Cells were fixed with formaldehyde for 10 min. The ChIP was performed with 25 µg of chromatin, 2 µg of ab8580 (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.
All batches of ab8580 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 - tri methyl K4 peptide (ab1342), indicating that this antibody specifically recognises the Histone H3 - tri methyl K4 modification.
John E. Mueller and J. Ruth German (Mary Bryk lab)
Rabbit polyclonal to Histone H3 tri methyl K9 (ab8580) at 1/5000 on S. cerevisiae whole cell lysate (40 ug per lane).
Protein resolved on 15% SDS-PAGE gel. After transfer to PVDF membrane, blots were blocked in 1X PBS, 0.1% Tween-20, and 5% milk. ab1791 was diluted in 5 ml blocking buffer at 1/5000. Blots plus primary antibodies were either incubated overnight at 4C or at RT for 2 hr. Blots were washed 6X for 10 min each in PBS with 0.1% Tween-20 before addition of secondary antibodies. Secondary antibodies were diluted 1/2,000 in blocking buffer and incubated with blots for 2 hr at RT. Secondary blots were washed 4X for 10 min each in PBS with 0.1% Tween-20 and 2X for 10 min each in PBS.Protein resolved on 15% SDS-PAGE gel. After transfer to PVDF membrane, blots were blocked in 1X PBS, 0.1% Tween-20, and 5% milk. ab1791 was diluted in 5 ml blocking buffer at 1/5000. Blots plus primary antibodies were either incubated overnight at 4C or at RT for 2 hr.
ab8580 staining cultured human primary fibroblasts by ICC. Cells were PFA fixed and permeabilized in TritonX100 and saponin prior to blocking with 1% BSA for 1 hour at RT. The primary antibody was diluted 1/100 and incubated with the sample for 16 hours at 4°C. A FITC-conjugated rabbit anti-rabbit IgG antibody was used as the secondary.
Staining (green) with the anti-trimethyl Lysine K4 of Histone H3 antibody (ab8580) shows ringing of regions that appear as nucleoplasmic holes. These represent the positions of splicing factor compartments, which are preferentially associated with active genes and highly acetylated histone H3.
The antibody, as expected, fails to stain heterochromatin (red).
Mouse zygotes stained with the Tri-Methyl K4 histone H3 antibody
(green) and DNA (blue). This modification is readily detected in the two
pronuclei of the zygote.
This image was kindly supplied as part of the review submitted by Dr Maria Elena Torres Padilla (University of Cambridge, UK).
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