title: Isolation of the mTOR complexes by affinity purification.
abstract: The mammalian Target Of Rapamycin (mTOR) protein is a central component of the essential and highly conserved signaling pathway that emerged as a critical effector in regulation of cell physiology. Biochemical studies defined mTOR as the protein kinase that exists at least in two distinct complexes. The first complex has been characterized as the nutrient-sensitive mTOR complex 1 that controls cell growth and cell size by regulating protein synthesis and autophagy. The second complex of mTOR has been defined as the component of growth factor signaling that functions as a major regulatory kinase of Akt/PKB. Here, we provide the detailed methods how to purify the functional complexes of mTOR by affinity purification. In the first part, we describe the purification of the distinct mTOR complexes by immunoprecipitation. Purification of the soluble mTOR complexes is explained in the second part of this chapter.
authors: Department of Molecular and Cellular Oncology, University of Texas M. D. Anderson Cancer Center, Houston, TX, USA. dsarbass@mdanderson.org
authors: Dos D
authors: dos D
authors: Sarbassov
authors: Olga
authors: O
authors: Bulgakova
authors: Rakhmet I
authors: RI
authors: Bersimbaev
authors: Tattym
authors: T
authors: Shaiken
mesh_terms: D000915:Antibody Affinity; D001343:Autophagy; D018929:Cell Culture Techniques; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D057809:HEK293 Cells; D006367:HeLa Cells; D006801:Humans; D047468:Immunoprecipitation / Q000379:methods*; D000076222:Mechanistic Target of Rapamycin Complex 1; D046912:Multiprotein Complexes; D011506:Proteins / Q000302:isolation & purification*; D058570:TOR Serine-Threonine Kinases; D014157:Transcription Factors / Q000302:isolation & purification*
chemical_list: C493245:CRTC2 protein, human; D046912:Multiprotein Complexes; D011506:Proteins; D014157:Transcription Factors; D000076222:Mechanistic Target of Rapamycin Complex 1; D058570:TOR Serine-Threonine Kinases