ChIP results obtained with the antibody directed against HDAC1
ChIP was performed with the antibody against HDAC1 (Cat. No. 25287) on sheared chromatin from 4,000,000 HeLa cells. 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 EIF4A2 and GAPDH promoters, used as positive controls, and for the MYOD1 gene and 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).

ChIP-seq results obtained with the antibody directed against HDAC1
ChIP was performed on sheared chromatin from 4,000,000 HeLa cells using 2 μg of the antibody against HDAC1 (Cat. No. 25287) as described above. The immunoprecipitated 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 1 Mb region of the X-chromosome (figure 2A and B) and in two regions surrounding the GAPDH and EIF4A2 positive control genes, respectively (figure 2C and D).

Determination of the antibody titer
To determine the titer of the antibody, an ELISA was performed using a serial dilution of antibody directed against HDAC1 (Cat. No. 25287), crude serum and flow through. The plates were coated with the peptide 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 1:75,000.

Western blot analysis using the antibody directed against HDAC1
Whole cell extracts (25 μg, lane 1) and nuclear extracts (25 μg, lane 2) from HeLa cells were analysed by Western blot using the antibody against HDAC1 (Cat. No. 25287) diluted 1:1,000 in TBS-Tween containing 5% skimmed milk. The position of the protein of interest is indicated on the right (expected size: 55 kDa); the marker (in kDa) is shown on the left.

Immunofluorescence using the antibody directed against HDAC1
HeLa cells were stained with the antibody against HDAC1 (Cat. No. 25287) and with DAPI. Cells were fixed with 4% formaldehyde for 10 minutes and blocked with PBS/TX-100 containing 5% normal goat serum and 1% BSA. The cells were immunofluorescently labelled with the HDAC1 antibody (left) diluted 1:500 in blocking solution followed by an anti-rabbit antibody conjugated to Alexa488. The middle panel shows staining of the nuclei with DAPI. A merge of the two stainings is shown on the right.

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