Assessing Peroxisomal Protein Interaction by Immunoprecipitation.
ACBD5
Co-immunoprecipitation
Membrane contact sites
Organelle interactions
Peroxisomes
Phosphorylation
Protein binding assay
Protein interaction
VAPB
Journal
Methods in molecular biology (Clifton, N.J.)
ISSN: 1940-6029
Titre abrégé: Methods Mol Biol
Pays: United States
ID NLM: 9214969
Informations de publication
Date de publication:
2023
2023
Historique:
entrez:
23
3
2023
pubmed:
24
3
2023
medline:
28
3
2023
Statut:
ppublish
Résumé
Organelles physically interact with each other via protein tethering complexes that bridge the opposing membranes. Organelle membrane contacts are highly dynamic, implying dynamism of the tethering complexes. Alterations in the binding of the tethering proteins can be assessed by immunoprecipitation. Antibody-conjugated beads allow for purification of the target protein with its binding partners, which can subsequently be examined by western blot analysis. We present immunoprecipitation methods and strategies to examine protein interaction domains, and for the identification of residues important for the regulation of the interaction, here focusing on phosphorylation. We use the peroxisomal membrane protein ACBD5 and its paralog ACBD4, which both bind ER membrane protein VAPB to mediate peroxisome-ER contacts, as example. However, this method can be applied to other peroxisomal and non-peroxisomal (membrane) proteins.
Identifiants
pubmed: 36952197
doi: 10.1007/978-1-0716-3048-8_24
doi:
Substances chimiques
Membrane Proteins
0
Types de publication
Journal Article
Research Support, Non-U.S. Gov't
Langues
eng
Sous-ensembles de citation
IM
Pagination
345-357Subventions
Organisme : Biotechnology and Biological Sciences Research Council
ID : BB/N01541X/1
Pays : United Kingdom
Organisme : Biotechnology and Biological Sciences Research Council
ID : BB/T002255/1
Pays : United Kingdom
Organisme : Medical Research Council
ID : MR/N0137941/1
Pays : United Kingdom
Informations de copyright
© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.
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