H3K4me2 polyclonal antibody - Classic
Polyclonal antibody raised in rabbit against histone H3 containing the dimethylated lysine 4 (H3K4me2), using a KLH-conjugated synthetic peptide.
| Lot | A936-0014P |
|---|---|
| Concentration | 1.2 µg/µl |
| Species reactivity | Human, Arabidopsis: positive. Other species: not tested. |
| Type | Polyclonal |
| Purity | Affinity purified polyclonal antibody. |
|---|---|
| Host | Rabbit |
| Storage Conditions | Store at -20°C; for long storage, store at -80°C. Avoid multiple freeze-thaw cycles. |
| Storage Buffer | PBS containing 0.05% azide and 0.05% ProClin 300. |
| Precautions | This product is for research use only. Not for use in diagnostic or therapeutic procedures. |
| Applications | Suggested dilution | References |
|---|---|---|
| ChIP/ChIP-seq * | 1 µg/ChIP | Fig 1, 2 |
| ELISA | 1:500 | Fig 3 |
| Dot Blotting | 1:20,000 | Fig 4 |
| Western Blotting | 1:1,000 | Fig 5 |
| Immunofluorescence | 1:5,000 | Fig 6 |
* Please note that of the optimal antibody amount per IP should be determined by the end-user. We recommend testing 1-5 µg per IP.
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Validation Data
Figure 1. ChIP-seq results obtained with the Diagenode antibody directed against H3K4me2
ChIP was performed as described above using 1 μg of the Diagenode antibody against H3K4me2 (Cat. No. pAb-035-050). The IP’d DNA was analysed on an Illumina Genome Analyzer. Library preparation, cluster generation and sequencing were performed according to the manufacturer’s instructions. The 36 bp tags were aligned to the human genome using the ELAND algorithm. Figure 2 shows the peak distribution along the complete X-chromosome (figure 2A) and in 3 chromosomal regions surrounding the GAPDH, c-fos and ACTB genes (figure 2B, C and D, respectively).
Figure 2 ChIP results obtained with the Diagenode antibody directed against H3K4me2
ChIP was performed with the Diagenode antibody against H3K4me2 (Cat. No. pAb-035-050) on sheared chromatin from 1 million HeLaS3 cells using the “Auto Histone ChIP-seq” kit. A titration of the antibody consisting of 1, 2, 5 and 10 μg per ChIP experiment was analysed. IgG (2 μg/IP) was used as negative IP control. Quantitative PCR was performed with primers for the promoter and coding region of the active GAPDH gene, the promoter of the active c-fos gene and for the coding region of the inactive TSH2B. Figure 1 shows the recovery, expressed as a % of input (the relative amount of immunoprecipitated DNA compared to input DNA after qPCR analysis).
Figure 3 Determination of the titer
To determine the titer, an ELISA was performed using a serial dilution of the Diagenode antibody directed against H3K4me2 (Cat. No. pAb-035-050), crude serum and flow through in antigen coated wells. The antigen used was a peptide containing the histone modification of interest. By plotting the absorbance against the antibody dilution (Figure 3), the titer of the antibody was estimated to be 1:12,600.
Cross reactivity test using the Diagenode antibody directed against H3K4me2
A Dot Blot analysis was performed to test the cross reactivity of the Diagenode antibody against H3K4me2 (Cat. No. pAb-035-050) with peptides containing other modifications of histone H3 and H4 and the unmodified H3K4 sequence. One hundred to 0.2 pmol of the respective peptides were spotted on a membrane. The antibody was used at a dilution of 1:20,000. Figure 4 shows a high specificity of the antibody for the modification of interest.
Figure 5 Western blot analysis using the Diagenode antibody directed against H3K4me2
Histone extracts of HeLa cells (15 μg) were analysed by Western blot using the Diagenode antibody against H3K4me2 (Cat. No. pAb-035-050) diluted 1:1,000 in TBS-Tween containing 5% skimmed milk. The position of the protein of interest is indicated on the right; the marker (in kDa) is shown on the left.
Applications
| ELISA Enzyme-linked immunosorbent assay. Read more |
| DB Dot blotting Read more |
| WB Western blot : The quality of antibodies used in this technique is crucial for correct and specific protein identification. Diagenode offers huge selection of highly sensitive and specific western blot-validated antibodies. Learn more about: Load... Read more |
| IF Immunofluorescence: Diagenode offers huge selection of highly sensitive antibodies validated in IF. Immunofluorescence using the Diagenode monoclonal antibody directed against CRISPR/Cas9 HeLa cells transfected with a Cas9 expression vector (... Read more |
| ChIP-seq (ab) Read more |
| ChIP-qPCR (ab) Read more |
Documents
| Datasheet H3K4me2 pAb-035-050 DATASHEET Datasheet description | Download |
| Antibodies you can trust POSTER Epigenetic research tools have evolved over time from endpoint PCR to qPCR to the analyses of lar... | Download |
| Epigenetic Antibodies Brochure BROCHURE More than in any other immuoprecipitation assays, quality antibodies are critical tools in many e... | Download |
Publications
How to properly cite this product in your workDiagenode strongly recommends using this: H3K4me2 polyclonal antibody - Classic (Diagenode Cat# C15410035 Lot# A936-0014P). Click here to copy to clipboard. Using our products in your publication? Let us know! |
The Wnt-Driven Mll1 Epigenome Regulates Salivary Gland and Head and Neck Cancer. |
Histone variant H2A.Z deposition and acetylation directs the canonical Notch signaling response. |
The Arabidopsis LDL1/2-HDA6 histone modification complex is functionally associated with CCA1/LHY in regulation of circadian clock genes. |
SETBP1 induces transcription of a network of development genes by acting as an epigenetic hub. |
A CLK3-HMGA2 Alternative Splicing Axis Impacts Human Hematopoietic Stem Cell Molecular Identity throughout Development. |
Overexpression of histone demethylase Fbxl10 leads to enhanced migration in mouse embryonic fibroblasts. |
A novel microscopy-based high-throughput screening method to identify proteins that regulate global histone modification levels. |
The H3K4me3 histone demethylase Fbxl10 is a regulator of chemokine expression, cellular morphology and the metabolome of fibroblasts |