Purification of Adipocyte Lipid-biding Protein from Human and Murine Cells

This chapter discusses two primary experimental procedures allowed for the preparation of adipocyte lipid-binding protein (ALBP) from either human or murine adipose cells. ALBP possesses two reduced sulfhydryl groups, one at the amino terminus and one buried within the ligand-binding domain. The readily accessible amino-terminal sulfhydryl group makes chromatography on activated-thiol Sepharose 4B an effective purification step. More importantly, isolation of ALBP necessitates the handling of large volumes of crude extracts containing dissolved lipid in sufficient quantity to make conventional chromatographic procedures unfeasible. Aqueously dispersed lipid is easily removed by chromatography on Lipidex 1000 resin. The removal of endogenous lipid is required to purify the protein and to perform in vitro binding assays. The availability of homogeneous ALBP and several assays to test its binding characteristics will allow for a detailed analysis of this protein.