NFYB Antibody - ChIP-seq Grade

Figure 1. ChIP results obtained with the Diagenode antibody directed against NFYB
ChIP assays were performed using HeLa cells, the Diagenode antibody against NFYB (Cat. No. C15410241) and optimized 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 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 AURKA and GAPDH promoters, used as positive controls, and for the 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 antibody directed against NFYB
ChIP was performed on sheared chromatin from 4 million HeLa cells using 2 μg of the Diagenode antibody against NFYB (Cat. No. C15410241) as described above. The IP’d DNA was subsequently analysed on an Illumina HiSeq. Library preparation, cluster generation and sequencing were performed according to the manufacturer’s instructions. The 51 bp tags were aligned to the human genome using the BWA algorithm. Figure 2 shows the enrichment along the complete sequence and a 2 Mb region of the human X chromosome (fig 2A and B), and in a two genomic regions surrounding the AURKA and GAPDH positive control genes.

Figure 3. Western blot analysis using the Diagenode antibody directed against NFYB
Nuclear extracts from Neuro2A (lane 1), C8D30 (lane 2), NIH3T3 (lane 3), Raw264.7 (lane 4) and C2C12 (lane 5) cells (30 µg) were analysed by Western blot using the Diagenode antibody against NFYB (cat. No. C15410241) diluted 1:1,000. The position of the protein of interest is indicated on the right; the marker (in kDa) is shown on the left.

Figure 4. Immunoprecipitation using the Diagenode antibody directed against NFYB
Immunoprecipitation was performed on whole cell extracts from 293T cells using 2.5 μg of the Diagenode antibody against NFYB (Cat. No. C15410241). An equal amount of rabbit IgG was used as a negative control. The immunoprecipitated NFYB protein was detected by western blot with the NFYB antibody diluted 1:1,000. The IP with the NFYB antibody and with the IgG negative control are shown in lane 3 and lane 2, respectively. Lane 1 shows the input (40 μg of 293T whole cell extract).

Figure 5. Immunofluorescence with the Diagenode antibody directed against NFYB
HeLa cells were fixed with formaldehyde and stained with the Diagenode antibody against NFYB (cat. C15410241) diluted 1:500 (left). The right picture shows costaining with the cytoskeleton marker Phalloidin.