TITLE: SARS-CoV-2 nsp3 Mac1 macrodomain TR-FRET Peptide displacement Assay
AUTHORS: Haim Barr, Noa Lahav

[DESCRIPTION]
This is a HTRF-based peptide displacement assay

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Experiment Concentrations (From Stock to Assay)
     ReagentStockLoaded into CombiFinal in assay plateUnitsHis-SARS COV2 MAC1183000 5012.5nM Substrate (Biotin-ADPr) 100000001600400nMDetection solution    Streptavidin-XL665 (SA-XL)10.250.125%MAb Anti-6HIS-Eu cryptate Gold1000.250.125%Assay buffer    HEPES pH=7.025025 25 mMNaCl200 20 20 mMBSA0.50.050.05%Tween 20 0.50.050.05%HTRF PPI Europium Detection Buffer1001010 %
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Compound Plate Design for Dose Response:
    Total Assay Volume: 16 µL
    Compounds Top Assay Concentration: 100 µM
    Dilution Factor: 3
    Dose Response Points: 10
    Number of Replicates: 2
    Backfill with DMSO: Yes

Compounds Plate Design for 2-Point Assay:
    Total Assay Volume: 16 µL
    Compounds Assay Concentration: 100 µM and 50µM
    Dilution Factor: 2
    Dose Response Points: 2
    Number of Replicates: 2
    Backfill with DMSO: Yes
 

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

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

Reagents
 

Detection Solution
 
MAC1 Buffer
 
HTRF PPI Europium Detection Buffer
 
Assay Buffer
 

SECTION: Prepare 384-well Plate
2. PRIME Multi-Drop Combi Tube Dispensing Cassette MAC1 Buffer 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 (This confirms there are no bubbles in the Dispensing Cassette).
SECTION: Prepare 384-well Plate
2.1. DISPENSE 4 µL to Columns 1 and 23 of assay plate
Note: These will represent the inhibitor control columns

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 the MAC1 Buffer discharged from the cassette.
SECTION: Prepare 384-well Plate
3. PRIME Multi-Drop Combi Tube Dispensing Cassette His-SARS COV2 MAC1 Enzyme 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 (This confirms there are no bubbles in the Dispensing Cassette).
SECTION: Prepare 384-well Plate
3.1. DISPENSE 4 µL 50 nanomolar (nM) to Columns 1 and 23 of assay plate

Note: 
 50 nanomolar (nM) is four times the final concentration for the assay. It will be diluted to be a final concentration of 12.5 nanomolar (nM) 
Column 2 and Column 24 are neutral control columns (Contain: Enzyme, Substrate, DMSO; no experimental 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 50 nanomolar (nM) discharged from the cassette.
SECTION: Prepare 384-well Plate
4. CENTRIFUGE 1500 rpm, 1 min  plate to remove bubbles
SECTION: Prepare 384-well Plate
5. INCUBATE plate for 15 min atRoom temperature 
SECTION: Prepare 384-well Plate
6. PRIME Multi-Drop Combi Tube Dispensing Cassette 1600 nanomolar (nM) 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 (This confirms there are no bubbles in the Dispensing Cassette).
SECTION: Prepare 384-well Plate
6.1. DISPENSE 4 µL 1600 nanomolar (nM) into Columns 1 through 23 and 24 (the full plate)

Note: 
 1600 nanomolar (nM) is four times the final concentration for the assay. It will be diluted to be a final concentration of 400 nanomolar (nM) 


SECTION: Prepare 384-well Plate
6.2. 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 1600 nanomolar (nM) discharged from the cassette.
SECTION: Prepare 384-well Plate
7. CENTRIFUGE 1500 rpm, 1 min  plate to remove bubbles
SECTION: Prepare 384-well Plate
8. 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.
SECTION: Prepare 384-well Plate
9. PRIME Multi-Drop Combi Tube Dispensing Cassette 0.25 % volume 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 (This confirms there are no bubbles in the Dispensing Cassette).

SECTION: Prepare 384-well Plate
9.1. DISPENSE 8 µL 0.25 % volume into full plate

Note: 
 0.25 % volume is two times the final concentration for the assay. It will be diluted to be a final concentration of 0.125 % volume 
SECTION: Prepare 384-well Plate
9.2. 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 1600 nanomolar (nM) discharged from the cassette.
SECTION: Prepare 384-well Plate
10. CENTRIFUGE 1500 rpm, 1 min  plate to remove bubbles
SECTION: Prepare 384-well Plate
11. INCUBATE plate for 60 min at Room temperature 

Recommended: Clean/Empty the Multi-Drop Combi Reagent Dispenser and Dispensing Cassette during this incubation step
SECTION: Reat Plate Fluorescence
12. READ and RECORD the plate Relative fluorescence units (RFU) via the "Mac1 Protocol" on the PHERAstar FS Control Software.