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Figure 1. ChIP results obtained with the Diagenode monoclonal antibody directed against NRF1 ChIP was performed using K562 cells, the Diagenode monoclonal antibody against NRF1 (Cat. No. C15200013) and optimized PCR primer sets for qPCR. ChIP was performed with the “iDeal ChIP-seq” kit (cat. No. C01010055), using sheared chromatin from 4 million cells. 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 BRCA1 and AURKA promoters, used as positive controls, and for the coding region of the inactive MYOD1 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).
Figure 2. ChIP-seq results obtained with the Diagenode monoclonal antibody directed against NRF1 ChIP was performed with 2 μg of the Diagenode antibody against NRF1 (Cat. No. C15200013) on sheared chromatin from 4 million K562 cells 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 700 kb region of chromosome 1 (figure 2A and B) and in two regions surrounding the BRCA1 and AURKA positive control genes (figure 2C and D).
Figure 3. Western blot analysis using the Diagenode monoclonal antibody directed against NRF1 Whole cell extracts from K562 cells were analysed by Western blot using the Diagenode antibody against NRF1 (Cat. No. C15200013) diluted 1:1,000 in TBST containing 5% milk powder. 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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