H3K4me2 Antibody

Figure 1. ChIP results obtained with the monoclonal antibody directed against H3K4me2
ChIP assays were performed using HeLa cells, the monoclonal antibody against H3K4me2 (cat. No. C15200151), and optimized PCR primer sets for qPCR. ChIP was performed on sheared chromatin from 1.6 million cells. A titration of the antibody, consisting of 1, 5, and 10 µg per ChIP experiment was analyzed. IgG (5 µg/IP) was used as a 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. Cut&Tag results obtained with the monoclonal antibody directed against H3K4me2
Cut&Tag was performed on 50,000 K562 cells using 1 µg of the monoclonal antibody against H3K4me2 (cat. No. C15200151), the pA-Tn5 transposase (C01070001), and the iDeal Cut&Tag kit (cat. No. C01070021). The libraries were subsequently analyzed on an Illumina NovaSeq sequencer (2x50 paired-end reads) according to the manufacturer’s instructions. The tags were aligned to the human genome (hg38) using the BWA algorithm. Figure 2 shows the peak distribution along the complete sequence and a 50 kb region of the X chromosome (Figure 2A and 2B), and in two genomic regions on chromosomes 12 and 7 (Figure 2C and 2D, respectively).

Figure 3. Cross reactivity of the monoclonal antibody directed against H3K4me2
To test the specificity an ELISA was performed using a serial dilution of the monoclonal antibody against H3K4me2 (cat. No. C15200151). The wells were coated with peptides containing the unmodified H3K4 as well as the mono-, di- and trimethylated H3K4 and the dimethylated H3K9. Figure 3 shows a high specificity of the antibody for the modification of interest.

Figure 4. Western blot analysis using the monoclonal antibody directed against H3K4me2
Histone extracts (15 µg) from HeLa cells were analyzed by Western blot using the monoclonal antibody against H3K4me2 (cat. No. C15200151) 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 5. Immunofluorescence using the monoclonal antibody directed against H3K4me2
HeLa cells were stained with the antibody against H3K4me2 (cat. No. C15200151) 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 labeled with the H3K4me2 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 stains is shown on the right.