Purification of caveolae by affinity two-phase partitioning using biotinylated antibodies and NeutrAvidin–dextran

Abstract

A new concept for affinity two-phase partitioning was tested. The partitioning was based on the interaction of target membranes with a primary antibody which, in turn, interacted with a biotinylated secondary antibody and NeutrAvidin–dextran in a poly(ethylene glycol)/dextran two-phase system. Caveolae selectively redistributed from the top phase to the NeutrAvidin–dextran-containing bottom phase by employing anti-caveolin as the primary antibody. This immunoaffinity approach was more selective than the established sucrose gradient centrifugation method and resulted in highly purified caveolae from Triton X-100-treated liver and lung plasma membranes. The same approach, employing other selective primary antibodies, should facilitate the purification also of other membrane fractions.