Anti-Histone H3 (tri methyl K4) antibody - ChIP Grade
| Name | Anti-Histone H3 (tri methyl K4) antibody - ChIP Grade |
|---|---|
| Supplier | Abcam |
| Catalog | ab8580 |
| Prices | $404.00 |
| Sizes | 50 µg |
| Host | Rabbit |
| Clonality | Polyclonal |
| Isotype | IgG |
| Applications | ChIP ChIP Array ICC/IF ChIP WB IHC-F IP ChIPseq FC ICC/IF ICC/IF IHC-P |
| Species Reactivities | Mouse, Rat, Rabbit, Human, Pig, Yeast, Xenopus, Arabidopsis thaliana, C. elegans, Drosophila, Zebrafish, Marmoset, Rice, Bovine, Deer, Plant, Mammalian |
| Antigen | Synthetic peptide corresponding to Human Histone H3 aa 1-100 (tri methyl K4) conjugated to Keyhole Limpet Haemocyanin (KLH) |
| Description | Rabbit Polyclonal |
| Gene | HIST3H3 |
| Conjugate | Unconjugated |
| Supplier Page | Shop |
Product images
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 a goat anti-rabbit AlexaFluor®488 secondary (ab150077) 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 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.ab1340 - Histone H3 - mono methyl K4ab1342 - Histone H3 - tri methyl K4ab1771 - Histone H3 - mono methyl K9ab1772 - Histone H3 - di methyl K9ab1773 - Histone H3 - tri methyl K9ab1780 - Histone H3 - mono methyl K27ab1781 - Histone H3 - di methyl K27ab1782 - Histone H3 - tri methyl K27ab7228 - Histone H3 - unmodifiedab7768 - Histone H3 - di methyl K4
Anti-Histone H3 (tri methyl K4) antibody - ChIP Grade (ab8580) at 1 µg/ml + Calf Thymus Histone Preparation Nuclear Lysate at 0.5 µgSecondaryGoat polyclonal to Rabbit IgG - H&L - Pre-Adsorbed (HRP) at 1/3000 dilutionPerformed under reducing conditions.
All lanes : Anti-Histone H3 (tri methyl K4) antibody - ChIP Grade (ab8580) at 1/5000 dilutionLane 1 : S. cerevisiae whole cell lysate with WTLane 2 : S. cerevisiae whole cell lysate with set1Lane 3 : S. cerevisiae whole cell lysate with bre2Lane 4 : S. cerevisiae whole cell lysate with sdc1Lane 5 : S. cerevisiae whole cell lysate with shg1Lane 6 : S. cerevisiae whole cell lysate with spp1Lane 7 : S. cerevisiae whole cell lysate with swd1Lane 8 : S. cerevisiae whole cell lysate with swd3Lysates/proteins at 40 µg per lane.Performed under reducing conditions.
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.See Abreview
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).
IF of primary cultures of bladder cancer which arise from urothelial cells to determine whether the alterations in chromatin during cancer development would be enhanced by the histone modification antibodies. Both cell types gave very weak staining with the trimethyl-K9-H3 antibody.
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).
Human female lymphoblast immunostained with ab8580 (1:100)(yellowish green) specific for histone H3 lysine 4 (H3-K4) trimethylation; the DNA is stained red with propidium iodide (PI).Note the inactive X chromosome (arrow) and pericentromeric heterochromatin are largely devoid of this modification.
IHC-P image of Histone H3 (tri methyl K4) staining with ab8580 on tissue sections from adult marmoset testis. The sections were subjected to heat-mediated antigen retrieval using Dako antigen retrieval solution. The sections were blocked with 5% milk for 30 minutes at 25°C and then incubated with ab8580 (1/100 dilution) for 16 hours at 4°C. The secondary used was an Alexa-Fluor 555 conjugated goat anti-rabbit polyclonal.See Abreview
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