TITLE: MERS-CoV 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 MERS-CoV 3C-like 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. 

Final Experiment Concentrations
   Final Assay (in plate)  Reagent ConcentrationUnitsMERS Mpro50nMSubstrate550nMHEPES pH=7.320mMNaCl50mMBSA0.1mg/mlTriton X-1000.01%TCEP1mM


[BEFORE_START]
Note: Inhibitor compounds stock concentration is 20 mM. Compounds are pre-dispensed into 384 plates and stored at -20 C until use.

[GUIDELINES]
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
2. 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
2.1. 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
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). 
Discard Assay Buffer discharged from the cassette.
SECTION: Prepare 384 Well Plate
3. PRIME with 100 nanomolar (nM) by Multi-Drop Combi Tube Dispensing Cassette 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).

SECTION: Prepare 384 Well Plate
3.1. DISPENSE 10 µL 100 nanomolar (nM) to Columns 2 through 22 and Column 24

Note: 
100 nanomolar (nM) is two times the final concentration for the assay. It is diluted to be a final concentration of 50 nanomolar (nM).
Column 2 and Column 24 are neutral control columns (Contain: Enzyme, Substrate, DMSO, no compounds)

SECTION: Prepare 384 Well Plate
3.2. EMPTY Multi-Drop Combi Tube Dispensing Cassette (by selecting the EMPTY button on the Combi Dispenser until the tubes of the cassette are emptied.) 
Discard the 100 nanomolar (nM) discharged from the cassette.
SECTION: Prepare 384 Well Plate
4. CENTRIFUGE 15000 rpm, 1 min plate to remove bubbles

SECTION: Prepare 384 Well Plate
5. INCUBATE plate for 15 min at Room temperature 
⚠ Make sure the plate is protected from light!
6. PRIME with Assay Buffer by Multi-Drop Combi Tube Dispensing Cassette by selecting the PRIME button on the Combi Dispenser until the tubes were filled completely. Then, EMPTY the Multi-Drop Combi Tube Dispensing Cassette.

7. PRIME with 1100 nanomolar (nM) by Multi-Drop Combi Tube Dispensing Cassette 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).
8. DISPENSE 10 µL into Columns 1 through 24 (the full plate)

Note: 
1100 nanomolar (nM) is two times the final concentration for the assay. It is diluted to be a final concentration of 50 nanomolar (nM)
9. CENTRIFUGE 15000 rpm, 1 min plate in plate centrifuge to remove bubbles
10. INCUBATE plate for 60 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
11. READ and RECORD the plate Relative fluorescence units (RFU) via the "MERS Protocol" on the PHERAstar FS Control Software.
 Software is a standard Flourescence Assay set for Optimal excitation wavelength 485 nm, emission wavelength 528 nm, and a Gain of 300.
 

 

SECTION: Prepare Reagents
1. PREPARE all of the reagents/buffers required for this experiment.