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

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 f...

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Detalles Bibliográficos
Autores principales: Nguyen-Tien, Dat, Suzuki, Takehiro, Kobayashi, Toshihiko, Toyama-Sorimachi, Noriko, Dohmae, Naoshi
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Elsevier 2022
Materias:
Protocol
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8983420/
https://www.ncbi.nlm.nih.gov/pubmed/35403001
http://dx.doi.org/10.1016/j.xpro.2022.101263
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author Nguyen-Tien, Dat
Suzuki, Takehiro
Kobayashi, Toshihiko
Toyama-Sorimachi, Noriko
Dohmae, Naoshi
author_facet Nguyen-Tien, Dat
Suzuki, Takehiro
Kobayashi, Toshihiko
Toyama-Sorimachi, Noriko
Dohmae, Naoshi
author_sort Nguyen-Tien, Dat
collection PubMed
description 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).
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spelling pubmed-89834202022-04-07 Identification of the interacting partners of a lysosomal membrane protein in living cells by BioID technique Nguyen-Tien, Dat Suzuki, Takehiro Kobayashi, Toshihiko Toyama-Sorimachi, Noriko Dohmae, Naoshi STAR Protoc Protocol 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). Elsevier 2022-03-31 /pmc/articles/PMC8983420/ /pubmed/35403001 http://dx.doi.org/10.1016/j.xpro.2022.101263 Text en © 2022 The Authors https://creativecommons.org/licenses/by/4.0/This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).
spellingShingle Protocol
Nguyen-Tien, Dat
Suzuki, Takehiro
Kobayashi, Toshihiko
Toyama-Sorimachi, Noriko
Dohmae, Naoshi
Identification of the interacting partners of a lysosomal membrane protein in living cells by BioID technique
title Identification of the interacting partners of a lysosomal membrane protein in living cells by BioID technique
title_full Identification of the interacting partners of a lysosomal membrane protein in living cells by BioID technique
title_fullStr Identification of the interacting partners of a lysosomal membrane protein in living cells by BioID technique
title_full_unstemmed Identification of the interacting partners of a lysosomal membrane protein in living cells by BioID technique
title_short Identification of the interacting partners of a lysosomal membrane protein in living cells by BioID technique
title_sort identification of the interacting partners of a lysosomal membrane protein in living cells by bioid technique
topic Protocol
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8983420/
https://www.ncbi.nlm.nih.gov/pubmed/35403001
http://dx.doi.org/10.1016/j.xpro.2022.101263
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