Biology Reference
In-Depth Information
CHAPTER 11
Affinity Purification of Protein
Complexes in C. elegans
Esther Zanin
*
, Julien Dumont
y
, Reto Gassmann
*
,
Iain Cheeseman
x
, Paul Maddox
z
, Shirin Bahmanyar
*
,
Ana Carvalho
*
, Sherry Niessen
{
, John R. Yates III
{
,
Karen Oegema
*
and Arshad Desai
*
*
Ludwig Institute for Cancer Research and Department of Cellular & Molecular Medicine, University of
California San Diego, La Jolla, California, USA
y
Institut Curie-UMR144, Paris, France
z
Institute for Research in Immunology and Cancer, Department of Pathology and Cell Biology, University of
Montreal, Montreal, Quebec, Canada
x
Whitehead Institute for Biomedical Research, MIT, Cambridge, MA, USA
{
The Skaggs Institute for Chemical Biology and Department of Chemical Physiology, The Center for
Physiological Proteomics, The Scripps Research Institute, La Jolla, California, USA
Abstract
I. Introduction
II. Generating Tools for Biochemistry
A. Generating a Polyclonal Antibody
B. Assessing Antibody Specificity by Immunoblotting after RNAi
C. Tandem Affinity Purification Tags
D. Introduction of Transgenes for Expression of Tagged Proteins
E. Validating the Functionality of the Transgene-Encoded Tagged Protein
III. The Optimal Starting Material for Protein Purification
A. Growing Worms in Large-Scale Liquid Culture
B. Unsynchronized Liquid Starter Culture
C. Monitoring Worm Cultures
D. Harvesting Worms from Liquid Cultures
E. Isolation of Embryos and Synchronization as Starved L1 Larvae
F. Seeding Synchronized Cultures Using Starved L1 Larvae
G. Freezing Adult Worms for Immunoprecipitation
H. Freezing Embryos for Immunoprecipitation
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