MERS-CoV 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 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
| A | B | C |
|---|---|---|
| Reagent | Concentration | Units |
| MERS Mpro | 50 | nM |
| MERS Substrate peptide | 550 | nM |
| HEPES pH=7.3 | 20 | mM |
| NaCl | 50 | mM |
| BSA | 0.1 | mg/ml |
| Triton X-100 | 0.01 | % (v/v) |
| TCEP | 1 | mM |
For more information, please see the Materials section.
Before start
Note: Inhibitor compounds stock concentration is 20 mM . Compounds are pre-dispensed into 384 plates and stored at -20 C until use.
Steps
Prepare Reagents
PREPARE all of the reagents/buffers required for this experiment.
| A | B | C | D | E |
|---|---|---|---|---|
| Reagent | Stock Concentration | Concentration Loaded into Combi | Final Concentration | Units |
| MERS-Cov Mpro Enzyme (original stock) | 507000 | 100 | 50 | nM |
| 20230221 MERS Mpro Enzyme | 478000 | 100 | 50 | nM |
| MERS Substrate | 750000 | 1100 | 550 | nM |
| Assay Buffer | ||||
| HEPES (pH 7.3) | 40 | 20 | 20 | mM |
| Sodium Chloride | 100 | 50 | 50 | mM |
| BSA | 10 | 0.1 | 0.1 | mg/mL |
| Triton X-100 | 10 | 0.01 | 0.01 | % by volume |
| TCEP | 1000 | 1 | 1 | mM |
For more information, please see the Materials sec
Prepare 384 Well Plate
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.
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 Assay Buffer discharged from the cassette.
PRIME with 100nanomolar (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).
DISPENSE 10µL 100nanomolar (nM) to Columns 2 through 22 and Column 24
Note:
100nanomolar (nM)is two times the final concentration for the assay. It is diluted to be a final concentration of50nanomolar (nM).- Column 2 and Column 24 are neutral control columns (Contain: Enzyme, Substrate, DMSO, no 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
100nanomolar (nM)discharged from the cassette.
CENTRIFUGE 15000rpm plate to remove bubbles
INCUBATE plate for 0h 15m 0s at Room temperature
⚠ Make sure the plate is protected from light!
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.
PRIME with 1100nanomolar (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).
DISPENSE 10µL into Columns 1 through 24 (the full plate)
Note:
1100nanomolar (nM)is two times the final concentration for the assay. It is diluted to be a final concentration of50nanomolar (nM)
CENTRIFUGE 15000rpm plate in plate centrifuge to remove bubbles
INCUBATE plate for 1h 0m 0s at Room temperature
⚠ Make sure the plate is protected from light!
Recommended: Clean the Multi-Drop Combi Reagent Dispenser during this incubation step
Read Plate Fluorescence
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.
Equipment
| Value | Label |
|---|---|
| PHERAstar FS | NAME |
| Microplate reader | TYPE |
| BMG LABTECH | BRAND |
| 0471B0001A | SKU |
