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Isolation of a caveolae-enriched fraction from rat lung by affinity partitioning and sucrose gradient centrifugation

2003; Elsevier BV; Volume: 313; Issue: 1 Linguagem: Inglês

10.1016/s0003-2697(02)00561-4

1096-0309

Parisa Abedinpour, Bengt Jergil,

Erythrocyte Function and Pathophysiology

Caveolae were isolated from rat lungs by a combination of affinity partitioning and sucrose gradient centrifugation. After homogenization of the lungs directly in a polyethylene glycol–dextran two-phase system and conventional phase partitioning, the polyethylene glycol-rich top phase was affinity partitioned with fresh bottom phase containing dextran-linked wheat-germ agglutinin. The lectin selectively attracted plasma membranes to the bottom phase. The isolated plasma membrane fraction was treated with Triton X-100 or, alternatively, sonicated before centrifugation in a stepwise sucrose gradient. Caveolin-enriched material collected at the 5/24% sucrose boundary. This material also contained 5′-nucleotidase activity and actin. Electron microscopy showed the material to consist of a homogeneous population of 50- to 100-nm vesicles. This purification protocol should allow the facile purification of caveolae also from other tissues, facilitating structural and functional studies.