VALIDATION IMAGES
Whole cell extracts (25 μg, lane 1) and nuclear extracts (25 μg, lane 2) from HeLa cells were analyzed by Western blot using HDAC1 Polyclonal Antibody (Cat. No. bs-53113R) 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.
To determine the titer of the antibody, an ELISA was performed using a serial dilution of Bioss antibody directed against HDAC1 Antibody (Cat. No. bs-53113R), 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, the titer of the antibody was estimated to be 1:75,000.
HeLa cells were stained with the Bioss antibody against HDAC1 Antibody (Cat. No. bs-53113R) and with DAPI. Cells were fixed with 4% formaldehyde for 10’ and blocked with PBS/TX-100 containing 5% normal goat serum and 1% BSA. The cells were immunofluorescently labeled with the HDAC1 antibody (left) diluted 1:500 in blocking solution followed by a secondary 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.
ChIP was performed on sheared chromatin from 4,000,000 HeLa cells using 2 μg of HDAC1 antibody (Cat. No. bs-53113R). The IP’d DNA was subsequently analyzed 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. The figure shows the peak distribution along the complete sequence and a 1 Mb region of the X-chromosome (figure A and B) and in two regions surrounding the GAPDH and EIF4A2 positive control genes, respectively (figure C and D).