SARS-CoV-2 Mpro fluorescence dose response
Haim Barr, Noa Lahav
Disclaimer
The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.
Abstract
This is a functional, biochemical assay used to identify treatments for viral infectious disease that target SARS-COV-2 Main Protease (MPro).
Utilizing direct enzyme activity measurement method od, the experiment was performed in a 384-well plate reading the fluorescence intensity. This assay tested the mode of action of inhibition.
It was developed at the Weizmann Institute of Science, as a part of the ASAP Drug Discovery Consortium.
Experiment Assay Concentrations
| A | B | C |
|---|---|---|
| Reagent | Final Assay Concentration | Units |
| SARS Mpro | 5 | nM |
| SARS Substrate | 375 | nM |
| HEPES (pH 7.3) | 20 | mM |
| NaCl | 50 | mM |
| Glycerol | 10 | % by volume |
| TWEEN 20 | 0.01 | % by volume |
| TCEP | 1 | mM |
For more information, please check out the "Materials" Section
Before start
Note: Inhibitor compounds stock concentration is 20 mM . Compounds are pre-dispensed into 384 plates and stored at -200˚C until use.
Steps
Prepare 384 Well Plate
PRIME Multi-Drop Combi Tube Dispensing Cassette with Assay Buffer by selecting the PRIME button on the Combi Dispenser until the tubes are filled completely.
DISPENSE 10µL to Columns 1 and 23 of assay plate
- Note: These will represent the inhibitor control columns (Contain: Substrate, Assay Buffer, DMSO; no experimental compounds )
EMPTY Multi-Drop Combi Tube Dispensing Cassette (by selecting the EMPTY button on the Combi Dispenser until the tubes of the cassette are emptied). Discard the Assay Buffer discharged from the cassette.
Prepare Reagents
PRIME Multi-Drop Combi Tube Dispensing Cassette with 10nanomolar (nM) by selecting the PRIME button on the Combi Dispenser until the tubes were filled completely.
- Note: Be sure to cycle dispensing several times on a a clean plate lid (This confirms there are no bubbles in the Dispensing Cassette).
DISPENSE 10µL 10nanomolar (nM) to Columns 2 through 22 and also Column 24.
Note:
10nanomolar (nM)is two times the final concentration for the assay. It is diluted to be a final concentration of5nanomolar (nM).- Column 2 and Column 24 are neutral control columns (Contain: Enzyme, Substrate, DMSO; no experimental compounds )
EMPTY Multi-Drop Combi Tube Dispensing Cassette (by selecting the EMPTY button on the Combi Dispenser until the tubes of the cassette are emptied). Discard the 10nanomolar (nM) discharged from the cassette.
CENTRIFUGE 15000rpm plate to remove bubbles
INCUBATE plate for 0h 15m 0s at Room temperature
PRIME Multi-Drop Combi Tube Dispensing Cassette with Assay Buffer by selecting the PRIME button on the Combi Dispenser until the tubes are filled completely. Then, EMPTY the Multi-Drop Combi Tube Dispensing Cassette (by selecting the EMPTY button on the Combi Dispenser until the tubes of the cassette are emptied). Discard the Assay Buffer discharged from the cassette.
PRIME Multi-Drop Combi Tube Dispensing Cassette with 750nanomolar (nM) by selecting the PRIME button on the Combi Dispenser until the tubes were filled completely.
- Note: Be sure to cycle dispensing several times on a a clean plate lid (This confirms there are no bubbles in the Dispensing Cassette).
DISPENSE 10µL 750nanomolar (nM) into Columns 1 through 24 (the full plate)
Note:
750nanomolar (nM)is two times the final concentration for the assay. It is diluted to be a final concentration of375nanomolar (nM)
CENTRIFUGE plate at 15000rpm in plate centrifuge to remove bubbles
INCUBATE plate atRoom temperature for 0h 30m 0s
⚠ Make sure the plate is protected from light!
Recommended: Clean/Empty the Multi-Drop Combi Reagent Dispenser and Dispensing Cassette during this incubation step
Read Plate Fluorescence
READ and RECORD the plate Relative fluorescence units (RFU) via the "SARS Endpoint protocol" on the PHERAstar FS Control Software .
