Ubiquitin activating enzyme e1

Assays were performed essentially as described [10] (link). Briefly, 2.5 µg of recombinant mononucleosomes were incubated in a 50 µl reaction buffer containing 50 mM Tris-HCl, pH 7.5, 100 mM NaCl, 10 mM MgCl₂, 1 µM ZnOAc, 1 mM DTT, 30 nM ubiquitin activating enzyme E1 (Boston Biochem), 1.5 µM ubiquitin conjugating enzyme UbcH5a (Boston Biochem), 4 µM RNF168 (1–113) or RING1B/BMI1 complex, 22 µM ubiquitin (Boston Biochem) and 3.33 mM ATP at 30°C for 4 h. The reaction was terminated by addition of SDS-PAGE loading buffer. Assays with free histones were carried out with 10 µM of H2A or H2AX and the reactions were incubated overnight at 30 C. The samples were boiled and loaded on 15% SDS-PAGE gels, transferred to a nitrocellulose membrane, probed using specific antibodies and detected as described above.

H2AZ-containing nucleosome core particle (NCP) was obtained from Active Motif ® (81072). MacroH2A octamer and 601 DNA (207 bp) were obtained from Colorado State University Histone Source. MacroH2A NCP was reconstituted by the step dilution method^{57 (link)} by mixing the 601 DNA with octamer in a 1:1.2 ratio. NCP formation was confirmed with 6% native TBE gels.
In vitro ubiquitination assays were performed using 2.5 μg of recombinant mononucleosomes. NCPs were incubated in a 50 μL reaction buffer containing 50 mM Tris-HCl, pH 7.5. 100 mM NaCl, 10 mM MgCl₂, 1 μM ZnOAc, 1 mM DTT, 30 nM ubiquitin-activating enzyme E1 (Boston Biochem), 1.5 μM ubiquitin-conjugating enzyme UbcH5a (Boston Biochem), 22  μM ubiquitin (Boston Biochem), 3.33 mM ATP and 4 μM RNF168 (1-113) at 30 °C for overnight. The reaction was stopped by adding 1× Laemmli sample buffer. ubiquitination was analyzed by western blot.