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

Abedinpour, Parisa LU and Jergil, Bengt LU (2003) In Analytical Biochemistry 313(1). p.1-8
Abstract
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... (More)
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. (Less)
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author
organization
publishing date
type
Contribution to journal
publication status
published
subject
in
Analytical Biochemistry
volume
313
issue
1
pages
1 - 8
publisher
Elsevier
external identifiers
  • pmid:12576051
  • wos:000181093100001
  • scopus:0037325633
ISSN
1096-0309
DOI
10.1016/S0003-2697(02)00561-4
language
English
LU publication?
yes
id
9b5ecba4-b19c-4735-88ed-4d7c1ab44142 (old id 124597)
date added to LUP
2007-06-26 08:16:04
date last changed
2018-01-07 05:35:00
@article{9b5ecba4-b19c-4735-88ed-4d7c1ab44142,
  abstract     = {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.},
  author       = {Abedinpour, Parisa and Jergil, Bengt},
  issn         = {1096-0309},
  language     = {eng},
  number       = {1},
  pages        = {1--8},
  publisher    = {Elsevier},
  series       = {Analytical Biochemistry},
  title        = {Isolation of a caveolae-enriched fraction from rat lung by affinity partitioning and sucrose gradient centrifugation},
  url          = {http://dx.doi.org/10.1016/S0003-2697(02)00561-4},
  volume       = {313},
  year         = {2003},
}