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Immunoprecipitation assay

SW Sugiko Watanabe
MI Makoto Iimori
DC David Virya Chan
EH Eiji Hara
HK Hiroyuki Kitao
YM Yoshihiko Maehara
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Nuclei were collected using swelling solution (5 mM PIPES, pH8.0, 85 mM KCl, 0.5% NP40), and nuclear chromatin-associated complexes were isolated using nuclear complex co-IP kit (Active motif #5400) according to the manufacturer’s protocols. Lysates were incubated with 30 μl Dynabeads Protein G (Thermo Fisher Scientific) and the indicated antibody complex for 4 hr at 4 °C. Beads were washed 5 times with TNE buffer (0.1% NP40, 50 mM Tris-HCl, pH 7.6, 150 mM NaCl, 5 mM EDTA). Thirty μl of Laemmli sample buffer was added to the beads to elute with heating at 96 °C for 5 min.

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