A Detergent-Free Method for Purifying Caveolae Membrane from Tissue Culture Cells

• Published in: Proceedings of the National Academy of Sciences - PNAS Vol. 92; no. 22; pp. 10104 - 10108
• Main Authors: Smart, Eric J., Ying, Yun-Shu, Mineo, Chieko, Richard G. W. Anderson
• Format: Journal Article
• Language: English
• Published: United States National Academy of Sciences of the United States of America 24.10.1995; National Acad Sciences; National Academy of Sciences
• Subjects: Antibodies; Biomarkers; Caveolae; Caveolins; Cell Fractionation - methods; Cell Membrane - ultrastructure; Cell membranes; Cells; Cells, Cultured; Cellular biology; Centrifugation; Cleaning; Cultured cells; Detergents; Electrophoresis, Polyacrylamide Gel; Endothelial cells; Enzymes - analysis; Fibroblasts - cytology; Fibroblasts - ultrastructure; Humans; Immunoblotting; Membranes; Microscopy, Electron; Organelles - ultrastructure; P branes; Proteins - analysis; Receptors; Skin - cytology; Skin - ultrastructure; Ultracentrifugation
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.92.22.10104

Current methods for purifying caveolae from tissue culture cells take advantage of the Triton X-100 insolubility of this membrane domain. To circumvent the use of detergents, we have developed a method that depends upon the unique buoyant density of caveolae membrane. The caveolae fractions that we obtain are highly enriched in caveolin. As a consequence we are able to identify caveolae-associated proteins that had previously gone undetected. Moreover, resident caveolae proteins that are soluble in Triton X-100 are retained during the isolation.