| Assay Method Information | |
| | MTase Filter-Binding Assay (FBA) |
| Description: | The transfer of tritiated methyl from [3H] SAM onto RNA substrate was monitored by filter-binding assay, performed according to the method described previously. (37) For hRNMT and SARS CoV-2 nsp14, assays were carried out in reaction mixture [40 mM Tris-HCl (pH 8.0), 1 mM DTT, 1 mM MgCl2, 2 μM SAM, and 0.1 μM 3H-SAM (Perkin Elmer)] in the presence of 0.7 μM GpppAC4 synthetic RNA and human RNA N7 MTase (hRNMT) (50 nM) and SARS-CoV-2 nsp14 (50 nM). For SARS CoV-2 nsp10/nsp16 (1.2 μM/0.2 μM) the reaction was performed in the presence of 0.7 μM mGpppAC4 synthetic RNA. For NS5 MTase (500 nM) the reaction buffer does not contain MgCl2 and the reaction was performed in the presence of 0.7 μM mGpppAC4 synthetic RNA. For vaccinia virus capping enzyme (D1–D12) (41 U), the commercial buffer (New England Biolabs) at 1× concentration was used and the reaction was performed in the presence of 0.7 μM GpppAC4 synthetic RNA. For vaccinia virus VP39 (24 U) the commercial buffer (New England Biolabs) at 1× concentration was used and the reaction was performed in the presence of 0.7 μM mGpppAC4 synthetic RNA. |
| Affinity data for this assay | |
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