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Figure 1. ChIP results obtained with the Diagenode antibody directed against OCT4 ChIP assays were performed using E14Tg2a mouse embryonic stem cells, the Diagenode antibody against OCT4 (Cat. No. C15410305) 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 known OCT4 targets UTF1, YES1 and ZSCAN10, used as positive control targets, and for the promoter of the GAPDH gene, used as a negative control. 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 OCT4 ChIP was performed on sheared chromatin from 4 million E14Tg2a mouse embryonic stem cells using 5 μg of the Diagenode antibody against OCT4 (Cat. No. C15410305) 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 mouse genome using the BWA algorithm. Figure 2 shows the peak distribution along the complete sequence of mouse chromosome 7 (figure 2A) and a 1Mb region containing the UTF1 positive control (figure 2B), and in a two genomic regions surrounding the YES1 and ZSCAN10 positive control genes (figure 2C and D).
Figure 3. Determination of the antibody titer To determine the titer of the antibody, an ELISA was performed using a serial dilution of the Diagenode antibody directed against OCT4 (Cat. No. C15410305). The plates were coated with the peptides used for immunization of the rabbit. By plotting the absorbance against the antibody dilution (Figure 2), the titer of the antibody was estimated to be 1:100,000.
Figure 4. Western blot analysis using the Diagenode antibody directed against OCT4 Nuclear extracts (15 μg) from human embryonic stem cells were analysed by Western blot using the Diagenode antibody against OCT4 (Cat. No. C15410305) 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.
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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