RESUMO
This chapter presents methods for exploiting the powerful tools available in the nematode worm Caenorhabditis elegans to understand the in vivo functions of cerebral cavernous malformation (CCM) genes and the organization of their associated signaling pathways. Included are methods for assessing phenotypes caused by loss-of-function mutations in the worm CCM genes kri-1 and ccm-3, CRISPR-based gene editing techniques, and protocols for conducting high-throughput forward genetic and small molecule screens.
Assuntos
Hemangioma Cavernoso do Sistema Nervoso Central/etiologia , Hemangioma Cavernoso do Sistema Nervoso Central/metabolismo , Fenótipo , Alelos , Animais , Sistemas CRISPR-Cas , Caenorhabditis elegans , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Edição de Genes , Predisposição Genética para Doença , Hemangioma Cavernoso do Sistema Nervoso Central/diagnóstico , Mutagênese , Mutação , Ribonucleoproteínas/metabolismoRESUMO
Affinity purification of a target protein followed by mass spectrometry of the purified peptides can be used to identify physical interactors of the protein of interest. Using this biochemical approach on proteins from whole organisms such as C. elegans can reveal novel in vivo protein interactions that cannot be identified using homology-based predictions or in vitro approaches. Here we describe affinity purification of a GFP-tagged target protein from whole worm lysates, digestion of the purified proteins into peptides, and preparation of the peptides for analysis by mass spectrometry. This protocol has been optimized for ChromoTek GFP-Trap® Magnetic Agarose beads, but it may be used with other tags and antibody-conjugated beads.
