Cells were harvested by trypsinization and washed in PBS, and 1 × 107 cells were resuspended in 1 ml of cell lysis buffer [5 mM Hepes-KOH (pH 8.0), 85 mM KCl, 0.4% (v/v) NP-40, 1× cocktail protease inhibitor, and 1 mM phenylmethylsulfonyl fluoride (PMSF)]. Cells were incubated on ice for 10 min and centrifuged at 6000g for 15 s at 4°C to pellet nuclei. After removal of the supernatant, nuclei were fixed at room temperature in the same volume of cell lysis buffer lacking NP-40 and containing 1% (v/v) formaldehyde. Fixed nuclei were incubated at room temperature for 10 min with mixing once by inversion during fixation. Cross-linking was quenched by addition of 75 μl of 1.5 M glycine and incubation for 5 min at room temperature, with two inversions during incubation. The mixture was centrifuged again at 6000g for 15 s at 4°C, and the supernatant was discarded. To the nuclei, 50 μl of nuclei lysis buffer [50 mM tris-HCl (pH 8.0), 1 mM EDTA, 1% (v/v) SDS, 1× CPI (cocktail protease inhibitor), and 1 mM PMSF] was added and incubated for 10 min on ice. The sample was then diluted by the addition of 750 μl of ice-cold buffer A [20 mM Hepes-KOH (pH 8.0), 2 mM MgCl2, 300 mM KCl, 1 mM EDTA, 10% (v/v) glycerol, and 0.1% (v/v) Triton X-100]. Chromatin was fragmented with Branson Digital Sonifier D250 at 70% duty cycle and level 3 output control for 10 × 30-s pulse with 60-s pause and was centrifuged at 14,000g for 10 min at 4°C to remove the solubilized chromatin within the supernatant. For IP, 200 μl of chromatin was added to 400 μl of ice-cold IP buffer [20 mM Hepes-KOH (pH 8.0), 150 mM KCl, 1.5 mM EDTA, 1% (v/v) Triton X-100, 1× CPI, and 1 mM PMSF] on ice, and antibody (5 and 10 μg per IP) was added as required. After 1 hour of rotation at 4°C, 30 μl of protein A/G beads (Santa Cruz Biotechnology) was added, and samples were rotated at 4°C overnight. The following day, beads were collected via centrifugation of samples for 30 s at 12,000g at room temperature, washed twice with 1 ml of room temperature buffer A and subsequently with 1 ml of TE [10 mM tris-HCl and 1 mM EDTA (pH 8.0)], and then centrifuged for 30 s at 12,000g at room temperature. Immunoprecipitated DNA was eluted from the protein A/G agarose beads by digestion with 200 μg of proteinase K in 100 μl of elution buffer [50 mM NaHCO3 and 1% (v/v) SDS], incubated at 56°C for 1 hour, purified with a QIAquick PCR Purification Kit (Qiagen), and eluted in TE. The purified DNA was dot-blotted on Bio-Dot SF (Bio-Rad Laboratories) according to the manufacturer’s protocol and detected by Southern blot using (TAACCC)6-Biotin probe or Alu probe, TGGCTCACGCCTGTAATCCCAGCACTTTGGGAGGCCGA-Biotin. The amount of DNA immunoprecipitated was normalized for each sample on the basis of the amount of input telomeric DNA, as determined by signal intensity from standard curves for each sample. The results were analyzed by ImageJ.

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