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Figure 1. ChIP results obtained with the Diagenode antibody directed against HDAC3 ChIP was performed with the Diagenode antibody against HDAC3 (Cat. No. C15410364) on sheared chromatin from 4,000,000 HeLa cells with the iDeal ChIP-seq kit for TF’s. An antibody titration consisting of 1, 2, 5 and 10 µg per ChIP experiment was analysed. IgG (2 µg/IP) was used as negative IP control. QPCR was performed with primers specific for the CDK4, FAM134A and HNRNPH2 promoters, used as positive controls, and for the MYOD1 gene, used as 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 HDAC3 ChIP was performed on sheared chromatin from 4,000,000 HeLa cells using 2 µg of the Diagenode antibody against HDAC3 (Cat. No. C15410364) as described above. The IP'd DNA was subsequently analysed on an Illumina HiSeq 4000. 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 Mb region of chromosome 3 (figure 2A and B) and in three genomic regions surrounding the CDK4, FAM134A and HNRNPH2 positive control genes, respectively (figure 2C, D and E).
Figure 3. Determination of the antibody titer To determine the titer of the antibody, an ELISA was performed using a serial dilution of Diagenode antibody directed against HDAC3 (Cat. No. C15410364). The plates were coated with the peptides used for immunization of the rabbit. By plotting the absorbance against the antibody dilution (Figure 3), the titer of the antibody was estimated to be 1:450,000.