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J Biol Chem, Vol. 274, Issue 33, 23144-23154, August 13, 1999
From the Department of Pathology, Harvard Medical School, Beth
Israel Deaconess Medical Center, Boston, Massachusetts 02215 and the
Sidney Kimmel Cancer Center, San Diego, California 92121
Defining the molecular composition of caveolae is
essential in establishing their molecular architecture and functions.
Here, we identify a high affinity monoclonal antibody that is specific for caveolin-1
Immunoisolation of Caveolae with High Affinity Antibody Binding
to the Oligomeric Caveolin Cage
TOWARD UNDERSTANDING THE BASIS OF PURIFICATION
and rapidly binds caveolin oligomerized around intact
caveolae. We use this antibody (i) to develop a new simplified method
for rapidly isolating caveolae from cell and tissue homogenates without
using the silica-coating technology and (ii) to analyze various
caveolae isolation techniques to understand how they work and why they
yield different compositions. Caveolae are immunoisolated from rat lung
plasma membrane fractions subjected to mechanical disruption.
Sonication of plasma membranes, isolated with or without silica
coating, releases caveolae along with other similarly buoyant microdomains and, therefore, requires immunoisolations to purify caveolae. Shearing of silica-coated plasma membranes provides a
homogeneous population of caveolae whose constituents (i) remain unchanged after immunoisolation, (ii) all fractionate bound to the
immunobeads, and (iii) appear equivalent to caveolae immunoisolated after sonication. The caveolae immunoisolated from different low density fractions are quite similar in molecular composition. They
contain a subset of key signaling molecules (i.e. G protein and endothelial nitric oxide synthase) and are markedly depleted in
glycosylphosphatidylinositol-anchored proteins, 
-actin, and angiotensin-converting enzyme. All caveolae isolated from the cell
surface of lung microvascular endothelium in vivo appear to
be coated with caveolin-1. Caveolin-1 and -2 can also exist in
these same caveolae. The isolation and analytical procedures as well as
the time-dependent dissociation of signaling molecules from
caveolae contribute to key compositional differences reported in the
literature for caveolae. This new, rapid, magnetic immunoisolation procedure provides a consistent preparation for use in the molecular analysis of caveolae.
Copyright © 1999 by The American Society for Biochemistry and Molecular Biology, Inc.
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