| Field | Specification |
|---|---|
| Assay Type | |
| Detection Instrument(s) | Recombinant SARS-CoV-2 Mpro; 2x Assay Buffer; 0.5M DTT; 0.5 mM Fluorogenic substrate |
| Detection Method | |
| Product Type | |
| Shipping | |
| Storage |
Background
SARS-CoV-2 Mpro Assay Kit Mpro of Severe acute respiratory syndrome Coronavirus 2 (SARS-CoV-2 Mpro, also referred to as SARS-CoV- 2 Main protease, SARS-CoV-2 3CL protease) plays an essential role in viral replication by processing the polyproteins 1ab at 11 cleavage sites. Inhibiting the activity of this enzyme would block viral replication, making it a promising target for anti-coronaviral therapeutic agents. Description The Aurora SARS-CoV-2 Mpro assay kit is a homogeneous FRET-based assay for screening Mpro inhibitors. The assay is fast and convenient, and requires just two steps. In the first step, the Mpro enzyme is preincubated with the compound for 30 minutes. The reaction is initiated by adding substrate solution at the second step. Fluorescent intensity is measured with a fluorescent plate reader at the excitation emission wavelengths of 340-360 nm wavelengths of 460-480 nm. and Item 2X Protease Assay Buffer 0.5 M DTT 5 mM Substrate Recombinant SARS-CoV-2 Mpro Black low binding 96 well plate Amount 20 ml 200 µl 10 µl 5 µg 1 Storage -20°C -20°C -80°C -80°C RT Materials supplied Catalogue Number 728205 728202 728206 Materials Needed but not supplied A microplate reader capable of measuring fluorescence at excitation wavelengths of 340-360 nm and emission wavelengths of 460-480 nm. Stability 12 months if stored under the indicated conditions.
Assay Protocol
- Step 1. Prepare the inhibitor compound solution. If the inhibitor compound is dissolved in water, make a solution of the compound 10-fold higher than the final concentration in 1X assay buffer (since you will add 5 µl to the 50 µl reaction). If the inhibitor compound is dissolved in DMSO, make a 100-fold higher concentration of the compound than the highest concentration you want to test in DMSO. Then make a 10-fold dilution in 1X assay buffer (at this step, the compound concentration is 10-fold higher than the final concentration and the DMSO concentration is 10%). To determine an IC50 or to test lower concentrations of the compound, prepare as series of further dilutions in 1X assay buffer containing 10% DMSO (the final concentration of the DMSO will be 1% in all samples).
- Step 2. Prepare Mpro solution. Thaw Mpro enzyme on ice. Upon first thaw, briefly spin tube to recover the full contents at the bottom of the tube. Make aliquots of the enzyme for single use. Store remaining undiluted enzyme at -80°C. Note: Mpro enzyme is sensitive to freeze/thaw cycles. Limit number freeze-thaw cycles for best results. Do not re-use the diluted enzyme. Dilute the Mpro enzyme to 5 ng/µl in 1X assay buffer. Add 20 µl of diluted enzyme solution to each of positive control well and inhibitor test well. Add 1X buffer to each of background well.
- Step 3. Add the inhibitor solution Add 5 µl of 1X assay buffer to each background well and positive control well if the inhibitor is diluted in 1X buffer. Add 5 µl of 1X assay buffer with 10% DMSO to each of background well and positive control well if the inhibitor is diluted in 1X assay buffer with 10% DMSO. Add 5 µl of diluted inhibitor solution from Step 2 to each of the inhibitor test well.
- Step 4. Incubate at room temperature for 30 minutes.
- Step 5. Prepare substrate solution During the incubation of the enzyme and the inhibitor solution, dilute the 5 mM substrate solution to 20 µM in 1X assay buffer. Make only enough solution as need for the assay. Store the remaining 5 mM Substrate solution to -80°C. Add 25 µl of diluted substrate solution to each of well, including background wells, positive control wells and the inhibitor test wells.
- Step 6. Incubate at room temperature for 2 hours.
- Step 7. Measure the fluorescent intensity Measure the fluorescent intensity at the excitation wavelengths of 340-360 nm and the emission wavelengths of 460-480 nm
Data Analysis
% Activity = (S − N) / (P − N) × 100S = sample signal | P = positive control (100%) | N = negative control (0%)
Calculate percentage activity of the enzyme % Activity= (Fp – Fb) – (Fi-Fb) Fp - Fb X 100 Where Fp refers to Fluorescent intensity of the positive control, Fb refers to Fluorescent intensity of background, and Fi refers to Fluorescent intensity of the inhibitor. Graph the percentage activity as a function of the inhibitor concentration to determine the IC50 of the test inhibitor. No CPD refers to no compound control (compound vehicle control).
Biological Pathway / Process
Viral Polyprotein Processing / Replication
Therapeutic / Disease Area
Infectious Disease (COVID-19)
▶▼What does this assay kit detect?
This kit detects SARS-CoV-2 Mpro / 3CL Protease (Main Protease) activity or binding using FRET (fluorogenic FRET substrate cleavage; Ex 340-360 nm / Em 460-480 nm). It is designed for cell-free, homogeneous conditions requiring no wash steps, and is compatible with high-throughput screening formats.
▶▼What instrument or plate reader is required?
A fluorescence microplate reader is required to read this assay. The specific excitation and emission wavelengths depend on the detection mode used. Please refer to the product datasheet for exact instrument settings and compatible reader models.
▶▼How many reactions are included, and is bulk ordering available?
This kit is available in 96 reactions. Each reaction is conducted in a 96-well / fluorescence plate reader format, making it directly compatible with standard liquid-handling robotics for HTS applications. For bulk orders or custom quantities, please contact BioHippo or submit a quote request — distributor pricing is available.
▶▼Has the assay performance been validated?
Yes. Aurora Biolabs validates each kit using reference compounds to confirm assay window, signal-to-background ratio, and reproducibility prior to release. Specific validation data are provided in the product datasheet. Users are encouraged to determine the Z′ factor under their own experimental conditions.
▶▼What are the storage and shipping requirements?
The kit ships on Dry Ice and should be stored at -80°C upon receipt. Individual components may have different storage requirements — please refer to the component table in the datasheet. Protein components are sensitive to freeze–thaw cycles; aliquot on first thaw and avoid repeated freeze–thaw.
Need a different assay kit format or extra support beyond the catalog item? We offer customization and add-on services for assay kits to better fit your target, workflow, and detection platform. Options may include assay format customization (biochemical, binding, or cell-based), detection method selection such as TR-FRET, fluorescence polarization, luminescence, or colorimetric readout, target-specific reagent development including recombinant proteins, conjugates, antibodies, substrates, tracers, and controls, as well as protocol optimization for sensitivity, specificity, signal window, and reproducibility. We can also support kit component adjustments, plate format and scale options, QC and validation packages, bulk or custom packaging, and related assay development services when a standard kit does not fully meet your needs. Click Talk to a Scientist to submit a request, email us at support@biohippo.com, or explore our Research Services for additional support. Our team will be in contact with you shortly.
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