Transport-specific fractionation for purification of ATP-dependent Ca²⁺ pumps

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.