ChIPAb+™ HDAC1 Antibody, rabbit polyclonal

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	The sheared cross-linked chromatin was immunoprecipitated (IP) using ChIP-validated antibodies to acetyl-histone H3, acetyl-histone H3- Lys9 (H3K9), trimethyl-histone H3-Lys9 (Upstate Biotechnology), HDAC1 and RNA Pol II (Santa Cruz Biotechnology). Each IP reaction consisted of 125 μl of chromatin + 375 μl of dilution buffer with protease inhibitors + 20 μl of protein G magnetic beads + 5 μg of primary antibody. The IP reactions were incubated at 4° C overnight with rotation. IgG from the same species as the primary antibodies served as negative controls. Magnetic beads were separated using a magnetic separator (Biolabs) and the supernatant was discarded. The Protein G magnetic beads-antibody-chromatin complex was incubated with a series of wash buffers provided in the Magna ChIP G tissue Kit: one time each for 5 min each wash on a rotating platform followed by magnetic clearance and careful removal of the supernatant fractions: 500 μl low salt immune complex wash buffer, 500 μl high salt immune complex wash buffer, 500 μl LiCl immune complex wash buffer, 500 μl TE buffer.

Protocol tips

The sheared cross-linked chromatin was immunoprecipitated (IP) using ChIP-validated antibodies to acetyl-histone H3, acetyl-histone H3- Lys9 (H3K9), trimethyl-histone H3-Lys9 (Upstate Biotechnology), HDAC1 and RNA Pol II (Santa Cruz Biotechnology). Each IP reaction consisted of 125 μl of chromatin + 375 μl of dilution buffer with protease inhibitors + 20 μl of protein G magnetic beads + 5 μg of primary antibody. The IP reactions were incubated at 4° C overnight with rotation. IgG from the same species as the primary antibodies served as negative controls. Magnetic beads were separated using a magnetic separator (Biolabs) and the supernatant was discarded. The Protein G magnetic beads-antibody-chromatin complex was incubated with a series of wash buffers provided in the Magna ChIP G tissue Kit: one time each for 5 min each wash on a rotating platform followed by magnetic clearance and careful removal of the supernatant fractions: 500 μl low salt immune complex wash buffer, 500 μl high salt immune complex wash buffer, 500 μl LiCl immune complex wash buffer, 500 μl TE buffer.

Publication protocol

Tumor samples that were snap frozen in liquid nitrogen and stored at −80°C were used for ChIP assays. MDA-MB-231 tumors from the treatment groups 1) vehicle control (VC), 2) KX-01, 3) tamoxifen (TAM), and 4) TAM + KX-01 were used. MCF-7 tumors were used as a positive control for ERα expression. The ChIP assay was performed using the Magna ChIP G tissue kit according to the manufacturer’s protocol (Millipore) and our previous studies (23). Briefly, a 5 mm3 tumor tissue piece was obtained using a micro-dissection punch and the sample was dispersed in 1 ml Magna ChIP G tissue stabilization solution with protease inhibitors and then cross-linked using 1% formaldehyde treatment (prepared fresh; 270 μl of 37% formaldehyde [Sigma] to 10 ml of PBS). Glycine (125 mM) was used to quench the formaldehyde and block further cross linking. After centrifugation at 800 × g at 4°C for 5 min, the pellet was rinsed in PBS, suspended in 500 μl Magna ChIP G tissue lysis buffer, vortexed well and incubated on ice for 15 min. Cells were then centrifuged at 800 × g at 4°C for 5 min and the supernatant was removed. The cell pellet was re-suspended in 125 μl Magna ChIP dilution buffer in a 1.5 ml tube, and the samples were sonicated using the Bioruptor automatic sonicator (Diagenode, Denville, NJ) at 4°C for 12 cycles of 30 seconds “ON”/30 seconds “OFF” to shear chromatin and generate DNA fragments of 200-1000 base pairs. 5 μl (1%) of the content was removed and saved in 4° C as input. The sheared cross-linked chromatin was immunoprecipitated (IP) using ChIP-validated antibodies to acetyl-histone H3, acetyl-histone H3- Lys9 (H3K9), trimethyl-histone H3-Lys9 (Upstate Biotechnology), HDAC1 and RNA Pol II (Santa Cruz Biotechnology). Each IP reaction consisted of 125 μl of chromatin + 375 μl of dilution buffer with protease inhibitors + 20 μl of protein G magnetic beads + 5 μg of primary antibody. The IP reactions were incubated at 4° C overnight with rotation. IgG from the same species as the primary antibodies served as negative controls. Magnetic beads were separated using a magnetic separator (Biolabs) and the supernatant was discarded. The Protein G magnetic beads-antibody-chromatin complex was incubated with a series of wash buffers provided in the Magna ChIP G tissue Kit: one time each for 5 min each wash on a rotating platform followed by magnetic clearance and careful removal of the supernatant fractions: 500 μl low salt immune complex wash buffer, 500 μl high salt immune complex wash buffer, 500 μl LiCl immune complex wash buffer, 500 μl TE buffer. Following immunoprecipitation, protein-DNA cross-links were reversed by adding 100 μl Magna Chip elution buffer with proteinase K and incubated at 62° C for 2 h with shaking followed by incubation at 95 ° C for 10 min. Samples were allowed to cool to room temperature and the magnetic beads were separated and supernatant was transferred to a new tube and DNA was purified using spin columns according to the manufactures protocol. ChIP-purified DNA was amplified by standard PCR using primers for the ERα promoter (sense, 5’-GAACCGTCCGCAGCTCAAGATC-3’; antisense, 5’GTCTGACCGTAGACCTGCGCGTTG-3’) yielding a 150 bp fragment using the following reactions conditions: 2 μl of ChIP purified DNA or 1% total input DNA, 200 nmol/L of each primer, 1.5 mmol/L MgCl2, 200 μmol/L dNTP, 10X PCR gold buffer (Applied Biosystems), and 2 units of Hot start AmpliTaq Gold DNA polymerase (Applied Biosystems) in a total volume of 20 μl. The reaction was initiated at 94°C for 4 min followed by 30 cycles of PCR (94°C, 30 s; 56°C, 30 s; 72°C, 1 min), and extended at 72°C for 5 min. After amplification, PCR products were separated on a 1.5% agarose gel and visualized by ethidium bromide staining using a Gel Doc 2000 instrument (Bio-Rad, Hercules, CA). All ChIP assays were performed three times yielding similar results.

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