Purification of caveolae by affinity two-phase partitioning using biotinylated antibodies and NeutrAvidin-dextran.
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.
Barinaga-Rementeria Ramírez
Irene
I
Biochemistry, Lund University, P.O. Box 124, SE-221 00 Lund, Sweden.
Abedinpour
Parisa
P
Jergil
Bengt
B
D000818:Animals; D000906:Antibodies / Q000737:chemistry*; D001360:Avidin / Q000737:chemistry*; D001710:Biotin / Q000737:chemistry*; D021941:Caveolae / Q000737:chemistry* / Q000648:ultrastructure; D003911:Dextrans / Q000737:chemistry*; D007163:Immunosorbent Techniques; D008099:Liver / Q000737:chemistry*; D008168:Lung / Q000737:chemistry*; D008297:Male; D017830:Octoxynol / Q000737:chemistry; D051381:Rats; D017207:Rats, Sprague-Dawley; D013395:Sucrose / Q000737:chemistry; D014461:Ultracentrifugation
D000906:Antibodies; D003911:Dextrans; C093483:neutravidin; D001360:Avidin; D013395:Sucrose; D001710:Biotin; D017830:Octoxynol