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Synthetic peptide conjugated to KLH derived from within residues 1 to the C-terminus Histone H3, mono methylated at R2.
Our Abpromise guarantee covers the use of ab15584 in the following tested applications.
The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
|IP||Use a concentration of 5 µg/ml.|
|ChIP/Chip||Use at an assay dependent concentration.|
|Dot Blot||Use at an assay dependent concentration.|
|IHC-P||1/80. Perform heat mediated antigen retrieval before commencing with IHC staining protocol.|
|PepArr||Use a concentration of 0.2 - 2 µg/ml.|
|Flow Cyt||Use at an assay dependent concentration.|
|WB||Use a concentration of 0.5 - 2 µg/ml. Detects a band of approximately 17 kDa (predicted molecular weight: 17 kDa).Can be blocked with Histone H3 (mono methyl R2) peptide (ab1775).|
|ChIP||Use 2-4 µg for 25 µg of chromatin.|
All batches of ab15584 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 - mono methyl R2 peptide (ab1775), indicating that this antibody specifically recognises the Histone H3 - mono methyl R2 modification.
1. ab1775 – Histone H3 - mono methyl R2 peptide
2. ab38488 – Histone H3 - symmetric di methyl R2
3. ab2853 – Histone H3 - asymmetric di methyl R2
4. ab17566 – Histone H3 - non-modified
5. ab33971 – Histone H3 - mono methyl R8
6. ab13845 – Histone H3 - mono methyl R17
7. ab16935 – Histone H3 - asymmetric di methyl R17
8. ab32948 – Histone H3 - symm
Image courtesy of Human Protein Atlas
ab15584 staining histone H3 mono methyl R2 in human testis, showing a distinct and strong nuclear staining pattern in ductus seminiferus and leydig cells. Paraffin embedded human skin tissue was incubated with ab15584 (1/80 dilution) for 30 mins at room temperature. Antigen retrieval was performed by heat induction in citrate buffer pH 6.
ab15584 was tested in a tissue microarray (TMA) containing a wide range of normal and cancer tissues as well as a cell microarray consisting of a range of commonly used, well characterised human cell lines. Further results for this antibody can be found at www.proteinatlas.org
ICC/IF image of ab15584 stained HeLa cells. The cells were 100% methanol fixed (5 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 (ab15584, 5µg/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.
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