| Title: |
Protocol for analyzing protein-protein interactions by split-luciferase complementation assays in human cell lysates |
| Authors: |
Claes, Zander; Lemaire, Sarah; Bollen, Mathieu |
| Source: |
ISSN:2666-1667 ; STAR Protocols, vol. 5 (4), Art.No. ARTN 103328. |
| Publisher Information: |
Cell Press |
| Publication Year: |
2024 |
| Subject Terms: |
Biochemical Research Methods; Biochemistry & Molecular Biology; CATALYTIC SUBUNIT; Life Sciences & Biomedicine; Science & Technology; SPECIFICITY; STRUCTURAL BASIS; Cell Biology; Molecular/Chemical Probes; Protein Biochemistry; Humans; HEK293 Cells; Luciferases; Protein Interaction Mapping; Protein Phosphatase 1; High-Throughput Screening Assays; Protein Binding |
| Description: |
Here, we present a lysate-based split-luciferase assay for examining protein-protein interactions (PPIs) in HEK293T cell lysates, exemplified by interactions between subunits of protein phosphatase PP1. We describe steps for storing and re-using lysates, sensor design, assay setup/optimization, and high-throughput screening of compound libraries. We then detail procedures for applying the assay as a research tool to characterize the dynamics of PPIs, which we illustrate with specific examples. For complete details on the use and execution of this protocol, please refer to Claes and Bollen.1. ; sponsorship: This project was financially supported by the Research Foundation-Flanders (FWO grant G095119N) , the KU Leuven Research Fund (BOF grant C14/20/101) , and the Foundation Against Cancer (STK) . (Research Foundation-Flanders (FWO)|G095119N, KU Leuven Research Fund (BOF grant)|C14/20/101, Foundation Against Cancer (STK)) ; status: Published |
| Document Type: |
article in journal/newspaper |
| File Description: |
application/pdf |
| Language: |
English |
| Relation: |
https://lirias.kuleuven.be/handle/20.500.12942/765471; https://doi.org/10.1016/j.xpro.2024.103328; https://pubmed.ncbi.nlm.nih.gov/39369386 |
| DOI: |
10.1016/j.xpro.2024.103328 |
| Availability: |
https://lirias.kuleuven.be/handle/20.500.12942/765471; https://hdl.handle.net/20.500.12942/765471; https://lirias.kuleuven.be/retrieve/263603f5-4f2c-4afb-94fa-923c5f5545cb; https://doi.org/10.1016/j.xpro.2024.103328; https://pubmed.ncbi.nlm.nih.gov/39369386 |
| Rights: |
info:eu-repo/semantics/openAccess ; public ; https://creativecommons.org/licenses/by-nc-nd/4.0/ |
| Accession Number: |
edsbas.98B0E13B |
| Database: |
BASE |