This chapter discusses transport-specific fractionation, which is a means for the identification of membrane transport proteins and as a purification procedure. The method depends on a specific biological property of a protein for isolation and employs a transport activity of the protein (rather than a ligand-binding property as would be used in affinity chromatography) as a physical tool for its purification. The most substantial success of this method in terms of a degree of purification has been its application by several laboratories to the purification of Ca2+-transport proteins, notably ATP-dependent Ca2+ pumps and the Na+/Ca2+ exchanger. The chapter discusses the methodology for the purification of reconstituted ATP-dependent Ca2+ pumps from mammalian brain synaptosomes. Two critical variables to consider when solubilizing the Ca2+-ATPase for use in transport-specific fractionation are the phospholipid-to-protein ratio and the cholate-to-phospholipid ratio.
ASJC Scopus subject areas
- Molecular Biology