Identification of the interacting partners of a lysosomal membrane protein in living cells by BioID technique

Abstract

Summary: The purpose of this protocol is to screen and identify the physiologically relevant interactors of a lysosomal protein in living cells. Here, we describe how to identify solute carrier family 15 member 4 (SLC15A4)-interacting proteins by BioID and mass spectrometry analysis. This protocol utilizes fusion of SLC15A4 with a mutant form of biotin ligase, BirA. The fusion protein can promiscuously biotinylate the proteins proximal to SLC15A4. The biotinylated endogenous proteins are pulled down by magnetic streptavidin beads and detected by mass spectrometry analysis.For complete details on the use and execution of this protocol, please refer to Kobayashi et al. (2021).

Keywords

• Cell-based Assays
• Molecular Biology
• Protein Biochemistry
• Proteomics
• Mass Spectrometry