For the deubiquitination assay, the purified PL pro (208 nM) was mixed with various concentrations of the compound (0-200 mM) in 20 mM Tris-HCl buffer (pH 6.8) before the substrate (400 nM), Ubiquitin-AMC (Enzo Life Sciences Inc., Farmingdale, NY), was added. All assays were performed at 37 C using the 96-well plate format. The enzyme activities were determined by monitoring the enhanced fluorescence emission upon substrate cleavage at excitation and emission wavelengths of 360 and 460 nm, respectively, on a SpectraMax M 2e Multimode Reader (Molecular Devices Co., Sunnyvale, CA). Release of AMC was measured in the same manner as that described above for the IC 50 measurements 25, 26 .
Spectramax m 2e multimode reader
Manufactured by Molecular Devices
Sourced in United States
The SpectraMax M 2e Multimode Reader is a versatile laboratory instrument that can perform a range of spectroscopic analyses. It is capable of measuring absorbance, fluorescence, and luminescence in microplates, cuvettes, and other sample formats. The SpectraMax M 2e offers a flexible configuration and can be equipped with various detection modes to accommodate a wide variety of assays and applications.
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4 protocols using spectramax m 2e multimode reader
1
Deubiquitination Assay for PL pro Inhibition
2
SARS-CoV-2 Protease Inhibition Assay
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SARS-CoV PL pro activity was measured by the method previously described by our group 9, 10, 22 . The inhibition assay was optimized in a 96-well plate to establish suitable assay conditions and incubation times. The fluorogenic peptide, Arg-Leu-Arg-Gly-Gly-AMC (ENZO Life Sciences, Farmingdale, NY) and 208 nM purified PL pro in 20 mM Tris-HCl buffer (pH 6.8) were used as the substrate and the enzyme, respectively. For the inhibition studies, 54 nM PL pro and 0-200 mM of the individual compounds were mixed with the substrate (50 mM) at 37 C, and the fluorescence intensity was monitored at excitation and emission wavelengths of 360 and 460 nm on a SpectraMax M 2e Multimode Reader (Molecular Devices Co., Sunnyvale, CA).
3
Quantification of OVA-specific IgG1 Antibodies
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OVA-specific immunoglobulin G1 (IgG1) antibodies were measured with a mouse OVA-IgG1 ELISA kit (Shibayagi, Japan) following the manufacturer's protocol. The absorption at 450 nm for OVA-specific IgG1 antibodies was measured with a SpectraMax M2e Multimode Reader (Molecular Devices).
4
Sialidase Inhibitory Assay Protocol
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The sialidase inhibitory assay was performed as reported previously (Kim et al., 2012 ▶ ; Cho et al., 2013 ▶ ). Briefly, 15 µl GH33 sialidase solution (0.1 U ml−1) was pre-mixed with 15 µl sample solution at different concentrations in 510 µl 50 mM sodium acetate buffer pH 5.0 in a cuvette. 60 µl 0.125 mM 4-methylumbelliferyl-α-d -N-acetylneuraminic acid sodium salt hydrate (Sigma, catalogue No. M8639) in buffer at pH 5.0 was then added to the mixture as a substrate to start the reaction at 310 K. 4-Methylumbelliferone was immediately quantified by fluorometric determination with a SpectraMax M2e Multimode Reader (Molecular Devices, USA). The excitation wavelength was 365 nm and the emission wavelength was 450 nm. Enzyme activity was recorded over a range of pre-incubation times (Fig. 1 ▶ a) or concentrations (Fig. 1 ▶ b). The data were analyzed using a nonlinear regression program (SigmaPlot; SPCC Inc., Chicago, Illinois, USA). The experimental enzyme activity was determined using the logistic curve represented by (1)
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