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Figure 1. ChIP results obtained with the Diagenode monoclonal antibody directed against H3.3 ChIP assays were performed using human HeLa cells, the Diagenode antibody against H3.3 (cat. No. C15210011) and optimized PCR primer sets for qPCR. ChIP was performed with the "iDeal ChIP-seq" kit (cat. No. C01010055) on sheared chromatin from 1,000,000 cells. A titration of the antibody consisting of 1, 2 and 5 µg per ChIP experiment was analysed. IgG (1 µg/IP) was used as negative IP control. QPCR was performed with primers for the coding regions of the active CCT5 and EIF2S3 genes, used as positive controls, and for the inactive MYOD1 and TSH2B genes, 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).
Figure 2.ChIP-seq results obtained with the Diagenode monoclonal antibody directed against H3.3 ChIP was performed with 1 µg of the Diagenode antibody against H3.3 (cat. No. C15210011) on sheared chromatin from 1,000,000 HeLa cells using the “iDeal ChIP-seq” kit as described above. The IP'd DNA was subsequently analysed on an Illumina HiSeq 2000. Library preparation, cluster generation and sequencing were performed according to the manufacturer's instructions. The 50 bp tags were aligned to the human genome using the BWA algorithm. Figure 2 shows the peak distribution along the complete sequence and a 3.5 Mb region of human chromosome 1 (figure 2A and B) and in two genomic regions surrounding the CCT5 and EIF2S3 positive control genes (figure 2C and D).
Figure 3. Western blot analysis using the Diagenode monoclonal antibody directed against H3.3 Western blot was performed on 1 µg of recombinant histone H3.1 (lane 2) and on 1 µg of recombinant histone H3.3 (lane 1) using the Diagenode monoclonal antibody against H3.3 (cat. No. C15210011) diluted 1:500 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.
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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