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Figure 1. ChIP results obtained with the Diagenode monoclonal antibody directed against H3K4me1 ChIP assays were performed using HeLa cells, the monoclonal antibody against H3K4me1 (Cat. No. MAb-150- 050) and optimized PCR primer sets for qPCR. Chromatin was sheared with the Diagenode Bioruptor using the “Shearing ChIP” kit (Cat. No. kch-redmod-100). ChIP was performed with the “OneDay ChIP” kit (Cat. No. kch-oneDIP-060), using sheared chromatin from 1.6 million cells. A titration of the antibody consisting of 1, 5 and 10 μg per ChIP experiment was analysed. IgG (5 μg/IP) was used as negative IP control. QPCR was performed with primers for the promoter and the coding region of the GAPDH gene, and for the RPL10 and HBB promoters. Figure 1 shows the recovery, expressed as a % of input (the relative amount of immunoprecipitated DNA compared to input DNA after qPCR analysis).
Figure 2. Cross reactivity of the Diagenode monoclonal antibody directed against H3K4me1 To test the specificity an ELISA was performed using a serial dilution of the Diagenode monoclonal antibody against H3K4me1 (Cat. No. MAb-150-050). The wells were coated with peptides containing the unmodified H3K4 as well as the mono-, di- and trimethylated H3K4 and the monomethylated H3K9. Figure 2 shows a high specificity of the antibody for the modification of interest.
Figure 3. Western blot analysis using the Diagenode monoclonal directed antibody against H3K4me1 Histone extracts (15 μg) from HeLa cells were analysed by Western blot using the Diagenode monoclonal antibody against H3K4me1 (Cat. No. MAb-150- 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.
Figure 4. Immunofluorescence using the Diagenode monoclonal antibody directed against H3K4me1 HeLa cells were stained with the Diagenode antibody against H3K4me1 (Cat. No. MAb-150-050) and with DAPI. Cells were fixed with 4% formaldehyde for 10’ and blocked with PBS/TX-100 containing 5% normal goat serum and 1% BSA. The cells were immunofluorescently labelled with the H3K4me1 antibody (left) diluted 1:500 in blocking solution followed by an anti-mouse antibody conjugated to Alexa594. The middle panel shows staining of the nuclei with DAPI. A merge of the two stainings is shown on the right.
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.
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