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In vitro ubiquitination assay
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For ubiquitination reactions, 3.2 μg of purified RNF168 was incubated with 0.35 μg purified E1 enzyme, 0.8 μg UbcH5c, 2.5 μg purified H2A-H2B and 2 μg purified ubiquitin in a buffer containing 25 mM Tris-HCl pH 7.4, 5 mM MgCl2, 100 mM NaCl, 1 μM DTT, 2 mM ATP, creatine kinase (1 U/ml) and creatine phosphate (10 mM) for 2 h at 30°C.
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