Functional analysis of detergent-solubilized and membrane-reconstituted ATP-binding cassette transporters.
ATP-binding cassette (ABC) transporters are vital to any living system and are involved in the translocation of a wide variety of substances, from ions and nutrients to high molecular weight proteins. This chapter describes methods used to purify and membrane reconstitute ABC transporters in a fully functional state. The procedures are largely based on our experience with substrate-binding protein-dependent ABC uptake systems from bacteria, but the approaches should be applicable to multisubunit membrane complexes in general. Also, we present simple methods, based on substrate binding or translocation, to follow the activity of the protein complexes in detergent-solubilized and/or membrane-reconstituted state(s).
Poolman
Bert
B
Department of Biochemistry, Groningen Biomolecular Sciences and Biotechnology Institute and Materials Science Centre(plus), University of Groningen, The Netherlands.
Doeven
Mark K
MK
Geertsma
Eric R
ER
Biemans-Oldehinkel
Esther
E
Konings
Wil N
WN
Rees
Douglas C
DC
D018528:ATP-Binding Cassette Transporters / Q000737:chemistry* / Q000302:isolation & purification* / Q000378:metabolism; D021961:Cell Membrane Structures / Q000737:chemistry*; D002845:Chromatography; D022162:Cytoplasmic Vesicles / Q000737:chemistry; D003902:Detergents / Q000737:chemistry*; D005453:Fluorescence; D008958:Models, Molecular; D012995:Solubility; D013057:Spectrum Analysis
D018528:ATP-Binding Cassette Transporters; D003902:Detergents