TITLE: SARS-COV-2 Main Protease (Mpro) Fluorescence Dose Response
AUTHORS: Haim Barr, Noa Lahav

[DESCRIPTION]
This is a functional, biochemical assay used to identify treatments for viral infectious disease in SARS-COV-2 Main Protease. 

Utilizing a direct enzyme activity measurement method, the experiment was performed in a 384-well plate reading the fluorescence intensity. This assay tested the mode of action of inhibition. 

[GUIDELINES]
Note: Inhibitor compounds stock concentration is 20 mM. Compounds are pre-dispensed into 384 plates and stored at -20 C until use.
    Plate Information:
        Total Assay Volume: 20 µL
        Compounds Top Assay Concentration: 100 µM
        Dilution Factor: 2
        Dose Response Points: 12
        Number of Replicates: 2
        Backfill with DMSO: Yes

[STEPS]
SECTION: Prepare 384 Well Plate
1. PRIME with Assay Buffer by Multi-Drop Combi Tube Dispensing Cassette by selecting the PRIME button on the Combi Dispenser until the tubes are filled completely.  


SECTION: Prepare 384 Well Plate
1.1. DISPENSE 10 µL to Columns 1 and 23 of assay plate
Note: These will represent the inhibitor control columns (Contain: Substrate, Assay Buffer, DMSO, no experimental compounds)
SECTION: Prepare 384 Well Plate
1.2. EMPTY Multi-Drop Combi Tube Dispensing Cassette 
SECTION: Prepare 384 Well Plate
2. PRIME with 10 nanomolar (nM)  by Multi-Drop Combi Tube Dispensing Cassette by selecting the PRIME button on the Combi Dispenser until the tubes are filled completely.  
Note: Be sure to cycle dispensing several times on a a clean plate lid




SECTION: Prepare 384 Well Plate
2.1. DISPENSE 10 µL 10 nanomolar (nM) to Columns 2 through 22 and Column 24

Note:
10 nanomolar (nM) is two times the final concentration for the assay. It is diluted to be a final concentration of 5 nanomolar (nM) 
Column 2 and Column 24 are neutral control columns (Contain: Enzyme, Substrate, DMSO, no compounds)




SECTION: Prepare 384 Well Plate
2.2. EMPTY Multi-Drop Combi Tube Dispensing Cassette (by selecting the EMPTY button on the Combi Dispenser until the tubes of the cassette are emptied.) 
Note: Discard the 10 nanomolar (nM) discharged from the cassette. 

SECTION: Prepare 384 Well Plate
3. CENTRIFUGE 15000 rpm, 1 min  the plate to remove bubbles


SECTION: Prepare 384 Well Plate
4. INCUBATE plate for 15 min at Room temperature 

5. PRIME with Assay Buffer by Multi-Drop Combi Tube Dispensing Cassette by selecting the PRIME button on the Combi Dispenser until the tubes are filled completely. Then, immediately EMPTY the Multi-Drop Combi Tube Dispensing Cassette.

6. PRIME with 750 nanomolar (nM)  by Multi-Drop Combi Tube Dispensing Cassette by selecting the PRIME button on the Combi Dispenser until the tubes are filled completely.  
Note: Be sure to cycle dispensing several times on a a clean plate lid
7. DISPENSE 10 µL 750 nanomolar (nM) to Columns 1 through 23 (the full plate)

Note:
 750 nanomolar (nM) is two times the final concentration for the assay. It is diluted to be a final concentration of 375 nanomolar (nM) 

8. CENTRIFUGE 15000 rpm, 1 min  the plate to remove bubbles
9. INCUBATE plate for 30 min at Room temperature 
⚠ Make sure the plate is protected from light

 Recommended: Clean the Multi-Drop Combi Reagent Dispenser during this incubation step
 

SECTION: Read Plate Fluorescence
10. READ and RECORD the plate Relative fluorescence units (RFU) via the "SARS Endpoint protocol" on the PHERAstar FS Control Software.