Methyltransferase activity of the NSP10-NSP16 complex and inhibitor screening was performed using the MTase-Glo assay (Cat. No. V7601, Promega) (Hsiao et al, 2016 ) according to the manufacturer’s instructions. In brief, 18.75 μM RNA cap-0 oligo (5’-(N7-MeGppp)ACAUUUGCUUCUGAC-3’) or no RNA as control was incubated in the presence of 740 nM co-purified NSP10-NSP16 protein complexes or no protein as control in reaction buffer (20 mM Tris-HCl pH 8.0, 50 mM NaCl, 1 mM EDTA, 3 mM MgCl2, 0.1 mg/ml BSA, 1 mM DTT) supplemented with 0.2 U/μL SUPERase RNAse inhibitor and 40 μM S-adenosylmethionine (SAM). NSP10-NSP16 complex was incubated with 100 μM inhibitor compound or DMSO as control for 10 min before adding the remaining components. Next, enzymatic reactions were kept for 2 h at 37 °C in 8 μl total volume in 384-well plates (CLS3824, Corning). After incubation, 2 μl of 5x MTase-Glo Reagent (Promega) was added to wells, plates were shaken for 1 min at 1000 rpm and further incubated for 30 min at RT. Then, 10 μl of MTase-Glo Detection Solution (Promega) was added, plates were again shaken for 1 min at 1000 rpm and further incubated for 30 min at RT. Finally, firefly luminescence intensity (~565 nm) was quantified in a SpectraMax iD5 microplate reader (Molecular Devices).
Cls3824
Manufactured by Corning
The CLS3824 is a lab equipment product manufactured by Corning. It is a device designed for scientific research and analysis applications. The core function of the CLS3824 is to facilitate the measurement and analysis of various samples and materials in a laboratory setting. No further details are available without the risk of making interpretations or extrapolations.
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2 protocols using cls3824
1
Inhibitor Screening of SARS-CoV-2 Methyltransferase
2
Methyltransferase Activity of NSP10-NSP16
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Methyltransferase activity of the NSP10-NSP16 complex and inhibitor screening was performed using the MTase-Glo assay (Cat. No. V7601, Promega) (Hsiao et al, 2016 (link)) according to the manufacturer’s instructions. In brief, 18.75 µM RNA cap-0 oligo (5’-(N7-MeGppp)ACAUUUGCUUCUGAC-3’) or no RNA as control was incubated in the presence of 740 nM co-purified NSP10-NSP16 protein complexes or no protein as control in reaction buffer (20 mM Tris-HCl pH 8.0, 50 mM NaCl, 1 mM EDTA, 3 mM MgCl2, 0.1 mg/ml BSA, 1 mM DTT) supplemented with 0.2 U/µL SUPERase RNAse inhibitor and 40 µM S-adenosylmethionine (SAM). The NSP10-NSP16 complex was incubated with 100 µM inhibitor compound or DMSO as a control for 10 min before adding the remaining components. Next, enzymatic reactions were kept for 2 h at 37 °C in 8 µl total volume in 384-well plates (CLS3824, Corning). After incubation, 2 µl of 5× MTase-Glo Reagent (Promega) was added to wells, plates were shaken for 1 min at 1000 rpm and further incubated for 30 min at RT. Then, 10 µl of MTase-Glo Detection Solution (Promega) was added, plates were again shaken for 1 min at 1000 rpm and further incubated for 30 min at RT. Finally, firefly luminescence intensity (~565 nm) was quantified in a SpectraMax iD5 microplate reader (Molecular Devices).
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