Affinity Purification of Protein Complexes in C. elegans - Caenorhabditis Elegans: Molecular Genetics and Development - page 294

the vectors and a detailed protocol for MosSCI can be found on the webpage of the
Jorgensen laboratory that developed this method ( http://sites.google.com/site/jor-
gensenmossci/Home ).

E. Validating the Functionality of the Transgene-Encoded Tagged Protein
Before using the tagged protein for biochemical studies it is important to validate
its functionality. The most straightforward test is whether the fusion protein can
rescue a mutant phenotype. If a mutant is not available, functional tests can be
conducted using RNAi. This requires that the endogenous messenger RNA is spe-
cifically targeted by a dsRNA. Re-encoding transgenes or using a different 3 0 UTR on
the transgene are two strategies that have been used for this purpose ( Audhya et al.,
2005; Dammermann et al., 2008 ). A detailed description of how we re-encode
transgenes was recently presented ( Green et al., 2008 ).

III. The Optimal Starting Material for Protein Purification

For proteins that are widely expressed, adult worms are the most straightforward
starting material to use since they are easily grown in large-scale using liquid culture.
If the protein of interest is enriched in embryos, embryonic extracts can be made by
bleaching adult hermaphrodites to isolate embryos. By carefully choosing the incu-
bation temperature it is also possible to enrich the embryonic extract either for young
( < 50 cells) or old embryos ( > 200 cells) (see Section III.I.). If the target protein is
present only in specific cell types, one can try to enrich for these cell types by using
conditional loss of function mutations. For example, by using a temperature-sensi-
tive mutant that does not form a germline one can enrich for somatic cells ( Beanan
and Strome, 1992 ). We describe in Section III.I. a strategy that uses a temperature-
sensitive meiotic arrest mutant to enrich for embryos in meiosis I. While flow
cytometry sorting methods have been developed to isolate specific cell types for
expression and small RNA analysis, the amounts enriched are not yet sufficient for
biochemical methods ( Cinar et al., 2005; Colosimo et al., 2004; Fernandez et al.,
2010; Zhang et al., 2002 ). Future miniaturization of protein isolation methods is
likely to enable proteomic characterization of individual cell types sorted by flow
cytometry.

A. Growing Worms in Large-Scale Liquid Culture
Worms are relatively straightforward to grow in biochemical quantities using
liquid cultures. Growing worms on egg plates is an alternative approach and a
detailed protocol for their use is described by Mains & McGhee ( Hope, 1999 ).
The following protocols describe growing worms in large-scale liquid culture to
obtain sufficient amount of either adult worms or embryos to perform