HDAC1 (D5C6U) XP® Rabbit mAb #34589

Protocol tips

Upstream tips	Protocol tips	Downstream tips
-It is highly critical that the chromatin is of appropriate size and concentration.	-For optimal ChIP and ChIP-seq results, use 10 μl of antibody and 10 μg of chromatin (approximately 4 x 106 cells) per IP. -It is important to keep the tissue cold to avoid protein degradation. -Use fresh formaldehyde that is not past the manufacturer's expiration date.	-Once in solution, store 1M DTT at -20°C.

Upstream tips
-It is highly critical that the chromatin is of appropriate size and concentration.
Protocol tips
-For optimal ChIP and ChIP-seq results, use 10 μl of antibody and 10 μg of chromatin (approximately 4 x 106 cells) per IP. -It is important to keep the tissue cold to avoid protein degradation. -Use fresh formaldehyde that is not past the manufacturer's expiration date.
Downstream tips
-Once in solution, store 1M DTT at -20°C.

Publication protocol

Cells were maintained in DMEM supplemented with 10% FBS. Approximately 5 × 107 cells in each group were cross-linked with formaldehyde (1% final concentration), washed with cold phosphate-buffered saline (PBS), lysed in buffer, and sonicated on ice to fragment the chromatin to the average length of 300 to 500 bp. The chromatin DNA was precipitated by either normal rabbit IgG (control) or polyclonal anti-MTA2 antibody (sc-28731; Santa Cruz Biotechnology, Santa Cruz, CA, USA). The protein–DNA complexes were incubated with protein A Sepharose beads, and eluted in 1% SDS/0.1 M NaHCO3. The protein–DNA cross-link was reversed by heating at 65 °C for 6 h. DNA was recovered by phenol–chloroform extraction and ethanol precipitation and then subjected to in-depth whole-genome DNA sequencing (CapitalBio Corporation, Beijing, China). The raw sequencing image data were examined by the Illumina analysis pipeline, aligned to the unmasked human reference genome (NCBI v36, hg18), and further analyzed by MACS (Model-based Analysis for ChIP-Seq). For the qChIP assay, anti-MTA2, anti-Snail (ab82846, Abcam, Cambridge, UK), anti-HDAC1 (34589, Cell Signaling Technology, Danvers, MA, USA), anti-H3Ac (06–599, Millipore, Bedford, MA, USA) and anti-H3 (ab1791, Abcam) antibodies were used, and the enrichment of the DNA template was analyzed by quantitative PCR. ChIP/Re-ChIP was done essentially the same as the primary ChIP. Bead elutes from the first immunoprecipitation were incubated with 10 mmol/L DTT and diluted 1:50 in dilution buffer (1% Triton X-100, 2 mmol/L EDTA, 150 mmol/L NaCl, and 20 mmol/L Tris-HCl (pH 8.1)) followed by Re-ChIP with the secondary antibodies.

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Manufacturer protocol

Download the product protocol from Cell Signaling Technology for HDAC1 (D5C6U) XP® Rabbit mAb #34589 below.

Download manufacturer protocol

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