Optimized protocol for in vivo affinity purification proteomics and biochemistry using C. elegans

Muriel Desbois; Joseph S Pak; Karla J Opperman; Andrew C Giles; Brock Grill

doi:10.1016/j.xpro.2023.102262

Optimized protocol for in vivo affinity purification proteomics and biochemistry using C. elegans

STAR Protoc. 2023 Jun 7;4(2):102262. doi: 10.1016/j.xpro.2023.102262. Online ahead of print.

Authors

Muriel Desbois¹, Joseph S Pak¹, Karla J Opperman¹, Andrew C Giles², Brock Grill³

Affiliations

¹ Center for Integrative Brain Research, Seattle Children's Research Institute, Seattle, WA 98101, USA.
² Division of Medical Sciences, University of Northern British Columbia, Prince George, BC V2N 4Z9 Canada.
³ Center for Integrative Brain Research, Seattle Children's Research Institute, Seattle, WA 98101, USA; Department of Pediatrics, University of Washington Medical School, Seattle, WA 98101, USA; Department of Pharmacology, University of Washington Medical School, Seattle, WA 98101, USA. Electronic address: brock.grill@seattlechildrens.org.

Abstract

We present an optimized protocol for in vivo affinity purification proteomics and biochemistry using the model organism C. elegans. We describe steps for target tagging, large-scale culture, affinity purification using a cryomill, mass spectrometry and validation of candidate binding proteins. Our approach has proven successful for identifying protein-protein interactions and signaling networks with verified functional relevance. Our protocol is also suitable for biochemical evaluation of protein-protein interactions in vivo. For complete details on the use and execution of this protocol, please refer to Crawley et al.,¹ Giles et al.,² and Desbois et al.³.

Keywords: Mass Spectrometry; Model Organisms; Neuroscience; Protein Biochemistry; Protein expression and purification; Proteomics.

Abstract

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