Human N-terminal His-tagged ubiquitin-like modifier activating enzyme 1 (UBA1) was expressed in E. coli BL21 RIL and purified via IMAC and size exclusion chromatography53 (link). The C-terminally His-tagged E2 enzyme UbcH7 was expressed in E. coli BL21 (DE3) and purified via IMAC54 (link). Ub was expressed in E. coli BL21 (DE3) and purified via IMAC and cation exchange chromatography7 (link).
The E2 charging assay was performed with 250 nM UBA1, 4 µM UbcH7, 15 µM Ub, 25 mM Tris·HCl (pH 7.5), 50 mM NaCl, 0.1 mM DTT, 10 mM MgCl2, and started with 0.2 mM of the indicated nucleotide (ATP, Ap3A, or Ap4A). For the SAP control, the nucleotides were pre-incubated with 0.5 units of shrimp alkaline phosphatase (rSAP) (New England Biolabs) at 37 °C for 30 min, followed by inactivation of the rSAP at 65 °C for 15 min. The reaction mixtures were incubated at 20 °C and enzymatic reactions were stopped at the indicated time points by the addition of 6× non-reducing SDS loading dye. To the DTT controls 1 µL of 1 M DTT was added before stopping the reaction. Samples were separated by 15% SDS-PAGE gels and stained with Coomassie blue.
The E2 charging assay was performed with 250 nM UBA1, 4 µM UbcH7, 15 µM Ub, 25 mM Tris·HCl (pH 7.5), 50 mM NaCl, 0.1 mM DTT, 10 mM MgCl2, and started with 0.2 mM of the indicated nucleotide (ATP, Ap3A, or Ap4A). For the SAP control, the nucleotides were pre-incubated with 0.5 units of shrimp alkaline phosphatase (rSAP) (New England Biolabs) at 37 °C for 30 min, followed by inactivation of the rSAP at 65 °C for 15 min. The reaction mixtures were incubated at 20 °C and enzymatic reactions were stopped at the indicated time points by the addition of 6× non-reducing SDS loading dye. To the DTT controls 1 µL of 1 M DTT was added before stopping the reaction. Samples were separated by 15% SDS-PAGE gels and stained with Coomassie blue.
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