{"doi":"10.17504/protocols.io.eq2ly7r2mlx9/v5","title":"SARS-CoV-2 nsp3 macrodomain Time-Resolved FRET peptide displacement assay v5","abstract":"This protocol details the Time-Resolved FRET (TR-FRET) assay for SARS-CoV-2 nsp3 (non-structural protein 3) macrodomain (Mac1) binding of adenosine diphosphate (ADP)–ribosylated (ADPr) peptide. This method measures the affinity of Mac1 to its potential inhibitors by using a specific ADPr-modified peptide that allows the detection of binding. When bound, the biotinylated-peptide and the HIS-tagged Mac1 form a proximity complex that is detected by TR-FRET using Streptavidin-Eu Cryptate and anti-HIS-XL665 as a donor/acceptor pair. Excitation of the Eu Cryptate complex at 325 nm emits a resonant energy of 625 nm which in turn excites the XL665 to emit fluorescence at 665 nm. This energy transfer occurs only when ADPr modified peptide is in sufficient proximity to Mac1 while inhibitors which displace the peptide will prevent energy transfer. Binding is reported as the ratio of Acceptor/Donor (Em/Em) X 10,000. ------------------------------------------------------- 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 % ------------------------------------------------------- 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 50 µM Dilution Factor: 2 Dose Response Points: 2 Number of Replicates: 2 Backfill with DMSO: Yes","journal":null,"year":2024,"id":508581,"datarank":0.0,"base_score":0.0,"endowment":0.0,"self_citation_contribution":0.0,"citation_network_contribution":0.0,"self_endowment_contribution":0.0,"citer_contribution":0.0,"corpus_percentile":null,"corpus_rank":null,"citation_count":0,"citer_count":0,"citers_with_citation_signal":0,"citers_with_endowment":0,"datacite_reuse_total":0,"is_dataset":false,"is_dataset_confidence":0.9579,"is_data_producer":false,"deposit_databanks":null,"is_oa":true,"file_count":0,"downloads":0,"has_version_chain":false,"published_date":"2024-01-01","fair_score":null,"fair_percentile":null,"algorithm_id":"datarank_citation_only_1hop_v6","ranking_scope":"data_only","authors":[{"id":1171213,"name":"Noa Lahav","orcid":null,"position":1,"is_corresponding":false},{"id":60987,"name":"Haim Barr","orcid":"0000-0001-9192-3983","position":0,"is_corresponding":true}],"reference_count":0,"raw_metadata":null,"created_at":"2026-07-19T02:11:10.049781Z","pmid":null,"pmcid":null,"fwci":null,"citation_percentile":null,"influential_citations":0,"oa_status":null,"license":null,"views":0,"total_file_size_bytes":0,"version_count":0,"fair_f":null,"fair_a":null,"fair_i":null,"fair_r":null,"fair_zscore":null,"fair_rationale":null,"fair_model":null,"fair_agent_version":null,"fair_fulltext_source":null,"fair_has_llm":null,"fair_computed_at":null,"clinical_trials":[],"software_tools":[],"db_accessions":[],"linked_datasets":[],"topics":[]}