Co-immunoprecipitation or pull-down assays are frequently used to analyze protein-protein interactions. In these experiments, western blotting is commonly used to detect prey proteins. However, sensitivity and quantification problems remain in this detection system. Recently, the HiBiT-tag-dependent NanoLuc luciferase system was developed as a highly sensitive detection system for small amounts of proteins. In this report, we introduce the method of using HiBiT technology for the detection of prey protein in a pull-down assay. Using this protocol, we demonstrate the formation of a ternary complex consisting of Japanese encephalitis virus NS4B and two host factors, namely valosin-containing protein, and nuclear protein localization protein 4, which is a critical biological event during flavivirus replication in cells.
Keywords: Flavivirus; HiBiT tag; Japanese encephalitis virus; Pull-down assay; Split nano luciferase.
Copyright © 2023 The Authors; This is an open access article under the CC BY-NC license (https://creativecommons.org/licenses/by-nc/4.0/).