ChIP results obtained with the monoclonal antibody directed against H3K4me3.
ChIP assays were performed using human HeLa cells, the monoclonal antibody against H3K4me3 (cat. No. 25256) and optimized PCR primer pairs for qPCR. ChIP was performed with the “Auto Histone ChIP-seq” kit, using sheared chromatin from 1 million cells on the SX-8G IP-Star automated system. A titration consisting of 1, 2, 5 and 10 µg of antibody per ChIP experiment was analyzed. IgG (2 µg/IP) was used as a negative IP control. Quantitative PCR was performed with primers specific for the promoter of the constitutively expressed GAPDH and c-fos genes, used aspositive controls, and for exon 2 of the inactive myoglobin (MB) gene and the Sat2 satellite repeat, used as negative controls. Figure 1 shows the recovery, expressed as a % of input (the relative amount of immunoprecipitated DNA compared to input DNA after qPCR analysis). These results are in accordance with the observation that trimethylation of K4 at histone H3 is associated with the promoters of active genes.
ChIP-seq results obtained with the monoclonal antibody directed against H3K4me3
ChIP was performed on sheared chromatin from 1 million HeLaS3 cells using 2 µg of the antibody against H3K4me3 (cat. No. 25256) as described above. The IP’d DNA was subsequently 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 two 5 Mb regions of chromosome 3 and 5 (figure 2A and B, respectively) and in two 100 kb regions surrounding the GAPDH and c-fos positive control genes (figure 2C and D). These results clearly show an enrichment of the H3K4 trimethylation at the promoters of active genes.
Cut&Tag results obtained with the monoclonal antibody directed against H3K4me3
CUT&TAG (Kaya-Okur, H.S., Nat Commun 10, 1930, 2019) was performed on 50,000 K562 cells using 1 µg of the monoclonal antibody against H3K4me3 (cat. No. 25256) and the pA-Tn5 transposase. The libraries were subsequently analysed on an Illumina NextSeq 500 sequencer (2x75 paired-end reads) according to the manufacturer’s instructions. The tags were aligned to the human genome (hg19) using the BWA algorithm. Figure 3 shows the peak distribution along the complete sequence and a 1.5 Mb zoomin of chromosome 1 (figure 3A and B, respectively).
Cross reactivity of the monoclonal antibody directed against H3K4me3
To test the specificity an ELISA was performed using a serial dilution of the monoclonal antibody against H3K4me3 (cat. No. 25256). The wells were coated with peptides containing the unmodified H3K4 as well as the mono-, di- and trimethylated H3K4 and the trimethylated H3K9. Figure 4 shows a high specificity of the antibody for the modification of interest.
Western blot analysis using the monoclonal antibody directed against H3K4me3
Histone extracts (15 µg) from HeLa cells were analysed by Western blot using the monoclonal antibody against H3K4me3 (cat. No. 25256) 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.
Immunofluorescence using the monoclonal antibody directed against H3K4me3
HeLa cells were stained with the antibody against H3K4me3 (cat. No. C15200152) 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 H3K4me3 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.
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