For the best experience on the Abcam website please upgrade to a modern browser such as Google Chrome.
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 dilution.|
|PepArr||Use a concentration of 0.2 - 2 µg/ml.|
|ICC||Use at an assay dependent dilution.|
|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).|
|IP||Use at an assay dependent dilution.|
|CHIPseq||Use at an assay dependent dilution. PubMed: 20952408|
|Flow Cyt||Use at an assay dependent dilution.|
|ICC/IF||1/100 - 1/5000.|
|IHC-P||Use at an assay dependent dilution. PubMed: 17634443|
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)
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).
Please note: All products are "FOR RESEARCH USE ONLY AND ARE NOT INTENDED FOR DIAGNOSTIC OR THERAPEUTIC USE"