Affinity Purification of Protein Complexes in C. elegans - Caenorhabditis Elegans: Molecular Genetics and Development - page 287

ABBREVIATIONS
APC, Anaphase-promoting complex; CBP, Calmodulin-binding peptide; ChIP,
Chromatin Immunoprecipitation; DMP, Dimethylpimelidimate; dsRNA, Double
stranded RNA; GST, Glutathione S-transferase; h, Hour; IP, Immunoprecipitation;
LAP, Localization and Affinity Purification; MosSCI, Mos1 mediated Single Copy
transgene Insertion; nAChR, Nicotinic acetylcholine receptor; RNAi, RNA inter-
ference; RT, Room temperature; TAP Tandem Affinity Purification; TEV, Tobacco
etch virus.
I. Introduction
Caenorhabditis elegans is widely recognized as a powerful model system for cell
and developmental biology. The landmark work that described the cell lineage from
embryo to adult provided the foundation to study cell biology in the context of
development in C. elegans ( Sulston et al., 1983 ). Research in C. elegans has
traditionally emphasized genetic and physiological approaches to elucidate gene
function. Classical epistasis analysis groups genes isolated by mutagenesis screens
into distinct pathways ( Huang and Sternberg, 2006 ). In the past decade, genome-
wide RNAi screens have greatly accelerated the annotation of gene functions
( Fernandez et al., 2005; Kamath et al., 2003; Piano et al., 2000, 2002;
Sonnichsen et al., 2005 ). Until recently, biochemical studies have lagged behind,
primarily due to the historical trajectory of C. elegans research. However,
C. elegans is a facile system for biochemical approaches as it is straightforward
to grow worms in large quantities, assess the functionality of tagged fusion proteins
using mutants or RNAi, and test the relevance of any identified interacting protein
rapidly through in silico analysis and in vivo methods ( Audhya and Desai, 2008;
Moresco et al., 2010 ).
In this chapter, we focus on methods in C. elegans for isolating protein complexes
and identifying new interacting proteins using mass spectrometry. In addition, we
describe cloning vectors that are useful for protein tagging and methods to assess
antibody specificity. To identify new interaction proteins we employ two major
strategies. In the first strategy, the target protein is purified using single-step
immunoprecipitation (IP) with an affinity purified polyclonal antibody and the
entire immunoprecipitate is subjected to mass spectrometric analysis.
Immunoprecipitation of the endogenous protein has several advantages: protein
expression is controlled by the endogenous promoter and protein function is not
altered by addition of a tag. The drawback of this approach is that a large number of
proteins are detected using current highly sensitive mass spectrometry methods.
This poses a challenge for discriminating between relevant and non-specific inter-
actions and therefore the significance of co-purified proteins needs to be carefully
evaluated in follow-up work. A potential additional drawback is that binding of the
primary antibody may block association with a subset of interacting components. In
the second strategy, we use tandem affinity purification (TAP) to isolate high