Apr 28, 2024
Version 4
MERS-CoV Mpro fluorescence dose response V.4
- Haim Barr1,2,
- Noa Lahav1,2
- 1The Weizmann Institute of Science;
- 2ASAP Discovery
- Haim Barr: General acknowledgement: The Wohl Drug Discovery institute, The Nancy and Stephen Grand Israel National Center for Personalized Medicine.;
Protocol Citation: Haim Barr, Noa Lahav 2024. MERS-CoV Mpro fluorescence dose response. protocols.io https://dx.doi.org/10.17504/protocols.io.eq2ly7r1rlx9/v4Version created by Mary-Ann Xavier
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: April 12, 2024
Last Modified: April 28, 2024
Protocol Integer ID: 98133
Funders Acknowledgement:
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)
Grant ID: U19AI171399
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.
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
Materials
Assay Buffer Reagents (Concentration listed are the stock concentrations)
- 40 millimolar (mM) HEPES Buffer (pH 7.3)Fisher ScientificCatalog #BP299-1 (or similar)
- 100 millimolar (mM) Sodium ChlorideFisher ScientificCatalog #S271 (or similar)
- 10 mg/mL BSA-Molecular Biology Grade - 12 mgNew England BiolabsCatalog #B9000S (or similar)
- 10 % volume Triton X-100Merck MilliporeSigma (Sigma-Aldrich)Catalog #T8787-50ML (or similar)
- 1000 millimolar (mM) TCEP HClP212121Catalog #SV-TCEP (or similar)
(all components are added fresh to the assay buffer before each experiment)
-------------------------------------------------------
Additional Reagents
- 507000 nanomolar (nM) MERS Mpro Enzyme*
*Note: The original MERS Mpro stock enzyme had a concentration of 507000 nanomolar (nM) but when a new stock solution was made/delivered on 2023-02-21the new stock had a concentration of 478000 nanomolar (nM) Both stock solutions were diluted with fresh assay buffer to create a 100 nanomolar (nM) solution before each experiment
- 750000 nanomolar (nM) MERS Substrate*
*Note: MERS Substrate (5-FAM)-GVLQSGLV-K(Dabcyl)-K-NH2 Stock was purchased from Peptide 2.0 and dissolved in DMSO with an original concentration of 750000 nanomolar (nM) however it was diluted with the same assay buffer used in the experiment before conducting each experiment to yield a concentration of 1100 nanomolar (nM)
Safety warnings
Please be sure to wear proper Personal Protective Equipment (PPE) while performing this experiment.
Before start
Note: Inhibitor compounds stock concentration is 20 mM. Compounds are pre-dispensed into 384 plates and stored at -20 C until use.
Prepare Reagents
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
Prepare 384 Well Plate
15m
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 10 µL Assay Buffer 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 100 nanomolar (nM) MERS MPro Enzyme 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 10 µL 100 nanomolar (nM) MERS MPro Enzyme to Columns 2 through 22 and Column 24
Note:
- 100 nanomolar (nM) MERS MPro Enzyme is two times the final concentration for the assay. It is diluted to be a final concentration of 50 nanomolar (nM) MERS MPro Enzyme .
- 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 100 nanomolar (nM) MERS MPro enzyme discharged from the cassette.
CENTRIFUGE 15000 rpm, Room temperature, 00:01:00 plate to remove bubbles
1m
INCUBATE plate for 00:15:00 at Room temperature
⚠ Make sure the plate is protected from light!
15m
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 1100 nanomolar (nM) MERS Substrate 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 10 µL 1100nM MERS Substrate into Columns 1 through 24 (the full plate)
Note:
- 1100 nanomolar (nM) MERS Substrate is two times the final concentration for the assay. It is diluted to be a final concentration of 50 nanomolar (nM) MERS Substrate
CENTRIFUGE 15000 rpm, Room temperature, 00:01:00 plate in plate centrifuge to remove bubbles
1m
INCUBATE plate for 01:00:00 at Room temperature
⚠ Make sure the plate is protected from light!
Recommended: Clean the Multi-Drop Combi Reagent Dispenser during this incubation step
1h
Read Plate Fluorescence
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
PHERAstar FS
NAME
Microplate reader
TYPE
BMG LABTECH
BRAND
0471B0001A
SKU
LINK
Expected result
Gain 300 should yield ~10,000 RFU in full reaction and ~6,000 RFU in Buffer Control