Analysis of in vivo protein complexes by coimmunoprecipitation from Caenorhabditis elegans.
Protein coimmunoprecipitation (co-IP) is a method used to analyze in vivo complex formation of various proteins. Although such an analysis supports the coexistence of proteins in a complex, a direct protein-protein interaction cannot be concluded unless further in vitro data are available. This protocol describes how to perform co-IPs from C. elegans whole-worm extracts using protein-specific antibodies. First, we describe how to culture a large number of worms while maintaining their overall appearance and wild-type fertility rates, which are important factors when analyzing the germline tissue. Next, we present a gentle and effective method to generate worm extracts with high protein concentrations that maintain protein complexes of high quality. Finally, we describe how to purify the protein of choice along with its associated complex members. The precipitated protein complex can be analyzed by either immunoblot analysis or mass spectrometry to identify the copurified protein components. When working with RNA-binding proteins, it is of interest to assess whether RNA molecules, rather than a direct interaction between the proteins, might mediate complex formation. For this purpose, an optional RNase digestion step to degrade the RNA in the extract is described.
Jedamzik
Britta
B
Max Planck Institute of Molecular Cell Biology and Genetics, 01307 Dresden, Germany.
Eckmann
Christian R
CR
D000818:Animals; D000906:Antibodies / Q000737:chemistry; D002021:Buffers; D017173:Caenorhabditis elegans / Q000502:physiology*; D029742:Caenorhabditis elegans Proteins / Q000737:chemistry; D015151:Immunoblotting; D047468:Immunoprecipitation / Q000379:methods*; D013058:Mass Spectrometry / Q000379:methods; D011485:Protein Binding; D011506:Proteins / Q000378:metabolism; D012313:RNA / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000737:chemistry; D011817:Rabbits; D012260:Ribonucleases / Q000378:metabolism
D000906:Antibodies; D002021:Buffers; D029742:Caenorhabditis elegans Proteins; D011506:Proteins; D016601:RNA-Binding Proteins; D012313:RNA; D012260:Ribonucleases