Affinity Purification of Protein Complexes in C. elegans - Caenorhabditis Elegans: Molecular Genetics and Development - page 286

I. Enriching for Specific Age Embryos
J. Enriching for Meiosis I Arrested Embryos
IV. Isolation of Nuclei from Embryos
V. Preparing Worm and Embryonic Extract
VI. Single-step Immunoprecipitation
A. Covalent Coupling of Antibodies to Protein A Beads
B. Immunoprecipitation
C. Sample Buffer Elution: For Silver Staining & Immunoblotting
D. Glycine Elution: For Mass Spectrometry
E. Urea Elution: For Mass Spectrometry
VII. Tandem Affinity Purification Using a LAP Tag
A. TEV Cleavage
B. S Protein Agarose
C. Sample Buffer Elution: For Silver Staining & Immunoblotting
D. Urea Elution: For Mass Spectrometry
VIII. Mass Spectrometry & Prioritization for Follow-up Experiments
IX. Summary
X. Solutions and Media
A. Worm Reagents
B. Large-Scale Liquid Culture
C. Isolation of Nuclei from Embryos
D. Single-step Immunoprecipitation
E. Tandem Affinity Purification using a LAP Tag
XI. Equipment
Acknowledgments
References
Abstract
C. elegans is a powerful metazoan model system to address fundamental questions
in cell and developmental biology. Research in C. elegans has traditionally focused
on genetic, physiological, and cell biological approaches. However, C. elegans is
also a facile system for biochemistry: worms are easy to grow in large quantities, the
functionality of tagged fusion proteins can be assessed using mutants or RNAi, and
the relevance of putative interaction partners can be rapidly tested in vivo.
Combining biochemistry with function-based genetic and RNA interference screens
can rapidly accelerate the delineation of protein networks and pathways in diverse
contexts. In this chapter, we focus on two strategies to identify protein-protein
interactions: single-step immunoprecipitation and tandem affinity purification.
We describe methods for growth of worms in large-scale liquid culture, preparation
of worm and embryo extracts, immunoprecipitation, and tandem affinity purifica-
tion. In addition, we describe methods to test specificity of antibodies, strategies
for optimizing starting material, and approaches to distinguish specific from non-
specific interactions.