title: Determination of Rab5 activity in the cell by effector pull-down assay.
abstract: Rab5 targets to early endosomes and is a master regulator of early endosome fusion and endocytosis in all eukaryotic cells. Like other GTPases, Rab5 functions as a molecular switch by alternating between GTP-bound and GDP-bound forms, with the former being biologically active via interactions with multiple effector proteins. Thus the Rab5-GTP level in the cell reflects Rab5 activity in promoting endosome fusion and endocytosis and is indicative of cellular endocytic activity. In this chapter, we describe a Rab5 activity assay by using GST fusion proteins with the Rab5 effectors such as Rabaptin-5, Rabenosyn-5, and EEA1 that specifically bind to GTP-bound Rab5. We compare the efficiencies of the three GST fusion proteins in the pull-down of mammalian and fungal Rab5 proteins.
authors: Key Laboratory of Biopesticides and Chemical Biology, Ministry of Education, Fujian Agriculture & Forestry University, 350002, Fuzhou, China.
authors: Yaoyao
authors: Y
authors: Qi
authors: Zhimin
authors: Z
authors: Liang
authors: Zonghua
authors: Z
authors: Wang
authors: Guodong
authors: G
authors: Lu
authors: Guangpu
authors: G
authors: Li
mesh_terms: D000818:Animals; D001681:Biological Assay / Q000379:methods*; D002460:Cell Line; D006224:Cricetinae; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D015151:Immunoblotting; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D033921:Vesicular Transport Proteins / Q000378:metabolism; D020696:rab5 GTP-Binding Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*
chemical_list: D011993:Recombinant Fusion Proteins; D033921:Vesicular Transport Proteins; D020696:rab5 GTP-Binding Proteins