Isolation of caveolae using affinity two-phase partitioning
(2004)- Abstract
- The intention of this work was to establish alternative purification methods to obtain highly purified caveolae from various tissues. In order to isolate caveolae, sufficiently pure plasma membranes are needed. A method is presented for the isolation of plasma membranes from lung tissue using a combination of conventional polyethylene glycol/dextran two-phase partitioning and affinity partitioning using the lectin wheat germ agglutinin as affinity ligand. A caveolae-enriched fraction was purified from lung plasma membranes isolated by this procedure. Caveolae were released from the membranes by Triton X-100 treatment or by sonication. Highly purified caveolae were obtained at low buoyant density by sucrose gradient centrifugation. The... (More)
- The intention of this work was to establish alternative purification methods to obtain highly purified caveolae from various tissues. In order to isolate caveolae, sufficiently pure plasma membranes are needed. A method is presented for the isolation of plasma membranes from lung tissue using a combination of conventional polyethylene glycol/dextran two-phase partitioning and affinity partitioning using the lectin wheat germ agglutinin as affinity ligand. A caveolae-enriched fraction was purified from lung plasma membranes isolated by this procedure. Caveolae were released from the membranes by Triton X-100 treatment or by sonication. Highly purified caveolae were obtained at low buoyant density by sucrose gradient centrifugation. The affinity method is advantageous to other methods for the isolation of plasma membranes and should be useful for other tissues as well.
A method to purify caveolae by immunoaffinity partitioning was developed exploiting the interaction between caveolin and anti-caveolin antibodies. A sandwich approach was used where primary antibodies directed against caveolin interacted with biotinylated secondary antibodies and NeutrAvidin coupled to dextran in a polyethylene glycol/dextran two-phase system. Caveolae were directed efficiently into the immunoaffinity bottom phase of the two-phase system by the anti-caveolin antibody. Immunoaffinity two-phase partitioning has wider applications potentially, as this technique should be useful to purify any type of membrane by selecting appropriate antibodies directed against surface components of the membrane of interest.
Caveolae were isolated to a high degree of purity from apical and basolateral domains of liver plasma membranes. The caveolae from the two domains were quite homogeneous as studied by immunoaffinity partitioning and electron microscopy. Analysis showed that the caveolae differed in properties in several respects. (Less) - Abstract (Swedish)
- Popular Abstract in Swedish
Målsättningen för detta arbete var att isolera caveolae för att kunna studera deras struktur och funktion. Caveolae är omegaformade inbuktningar i cellmembranet, ungefär 50-150 nm i diameter. Caveolae finns i många typer of celler, men inte i röda och vita blodkroppar eller nervceller. Man tror att de har flera viktiga funktioner. Exempelvis kan caveolae transportera molekyler mellan olika regioner av cellmembranet och även till membraner inne i cellen. Caveolae är också ett viktigt element i överföringen av signaler från cellens omgivning till dess inre. Än så länge vet man inte om caveolae utför liknande funktioner i olika organ i kroppen eller om funktionerna skiljer sig åt. Den vanliga... (More) - Popular Abstract in Swedish
Målsättningen för detta arbete var att isolera caveolae för att kunna studera deras struktur och funktion. Caveolae är omegaformade inbuktningar i cellmembranet, ungefär 50-150 nm i diameter. Caveolae finns i många typer of celler, men inte i röda och vita blodkroppar eller nervceller. Man tror att de har flera viktiga funktioner. Exempelvis kan caveolae transportera molekyler mellan olika regioner av cellmembranet och även till membraner inne i cellen. Caveolae är också ett viktigt element i överföringen av signaler från cellens omgivning till dess inre. Än så länge vet man inte om caveolae utför liknande funktioner i olika organ i kroppen eller om funktionerna skiljer sig åt. Den vanliga isoleringsmetoden för caveolae är baserad på att lösgöra dem från isolerade cellmembraner och sedan rena dem genom centrifugering i täthetsgradienter. Caveolae karakteriseras med hjälp av caveolin som är ett protein som finns i caveolaemembranet. Kolesterol och sfingolipider är viktiga lipider i detta membran, vilket gör det olösligt i vissa detergenter. Detta utnyttjas för att lösa ut caveolae från andra delar av cellmembranet.
Isolerade cellmembraner används som startmaterial för att rena caveolae. Här har en alternativ metod använts som går ut på att rena membranerna i s.k. tvåfassystem. Ett tvåfassystem består av två polymerlösningar i vatten som inte är blandbara. Dessa lösningar bildar två faser ungefär som olja och vatten. Olika membraner fördelar sig olika i ett sådant tvåfassystem och kan separeras från varandra på det sättet. För att förbättra reningen av cell membraner har ett sockerbindande protein, ett s.k. lektin som isolerats från vetegroddar, kopplats till den ena faspolymeren. Eftersom lektiner binder till sockerrester som finns på ytan av cellmembranet styr man cellmembranet till den fas som innehåller lektinet. Proceduren kallas affinitetsrening. Caveolae kan sedan lösas ut från cellmembranet med detergenter. För att rena caveolae har en ny metod utarbetats som också bygger på tvåfasseparation och affinitetsfördelning. Här används antikroppar som känner igen caveolinet i caveolae för att styra caveolae till en av faserna i tvåfassystemet. Affinitetsfördelning är en bra och snabb metod för rening av caveolae. Metoden kan också utnyttjas för isolering av andra membraner genom att byta till antikroppar som är riktade mot ytproteiner i dessa membraner.
Vi har alltså utvecklat alternativa tekniker för att isolera cellmembraner och caveolae för att kunna studera caveolaes exakta roll i celler. Exempelvis kan man studera sådana transportmekanismer i caveolae som utnyttjas av malaria- och sömnsjukeparasiter för att ta sig in i cellen. Även koleratoxin tas upp på samma sätt. Det är därför av intresse att rena caveolae för att kunna studera deras funktioner och därmed försöka lära sig styra sådana transportmekanismer. (Less)
Please use this url to cite or link to this publication:
https://lup.lub.lu.se/record/466695
- author
- Abedinpour, Parisa LU
- supervisor
- opponent
-
- Professor Strålfors, P., Hälsouniversitet, Linköping
- organization
- publishing date
- 2004
- type
- Thesis
- publication status
- published
- subject
- keywords
- metabolism, Biokemi, Metabolism, Biochemistry, basolateral, apical, immunoaffinity, wheat germ agglutinin, caveolin, caveolae caveolae enriched fraction, plasma membranes, rat lung, rat liver, Affinity Partitioning, two-phase partitioning
- pages
- 108 pages
- publisher
- Biochemistry, Center for Chemistry and Chemical Engineering, Lund University
- defense location
- Center for Chemistry and chemical engineering, Sölvegatan 41 sal B
- defense date
- 2004-02-27 10:30:00
- ISBN
- 91-7422-046-2
- language
- English
- LU publication?
- yes
- additional info
- Article: I. Isolation of a caveolae-enriched fraction from rat lung by affinity partitioning and sucrose gradient centrifugation1Parisa Abedinpour and Bengt JergilAnalytical Biochemistry 313 (2003) 1-8 Article: II. Purification of caveolae by affinity two-phase partitioning using biotinylated antibodies and NeutrAvidin-dextranIrene Barinaga Rementeria Ramírez, Parisa Abedinpour and Bengt JergilSubmitted to Analytical Biochemistry Article: III. Caveolae isolated from the apical and basolateral domain of rat liver differ in propertiesParisa Abedinpour and Bengt JergilManuscript
- id
- bfcf18ef-d362-484f-b571-af594364fe00 (old id 466695)
- date added to LUP
- 2016-04-04 11:39:03
- date last changed
- 2018-11-21 21:06:14
@phdthesis{bfcf18ef-d362-484f-b571-af594364fe00,
abstract = {{The intention of this work was to establish alternative purification methods to obtain highly purified caveolae from various tissues. In order to isolate caveolae, sufficiently pure plasma membranes are needed. A method is presented for the isolation of plasma membranes from lung tissue using a combination of conventional polyethylene glycol/dextran two-phase partitioning and affinity partitioning using the lectin wheat germ agglutinin as affinity ligand. A caveolae-enriched fraction was purified from lung plasma membranes isolated by this procedure. Caveolae were released from the membranes by Triton X-100 treatment or by sonication. Highly purified caveolae were obtained at low buoyant density by sucrose gradient centrifugation. The affinity method is advantageous to other methods for the isolation of plasma membranes and should be useful for other tissues as well.<br/><br>
<br/><br>
A method to purify caveolae by immunoaffinity partitioning was developed exploiting the interaction between caveolin and anti-caveolin antibodies. A sandwich approach was used where primary antibodies directed against caveolin interacted with biotinylated secondary antibodies and NeutrAvidin coupled to dextran in a polyethylene glycol/dextran two-phase system. Caveolae were directed efficiently into the immunoaffinity bottom phase of the two-phase system by the anti-caveolin antibody. Immunoaffinity two-phase partitioning has wider applications potentially, as this technique should be useful to purify any type of membrane by selecting appropriate antibodies directed against surface components of the membrane of interest.<br/><br>
<br/><br>
Caveolae were isolated to a high degree of purity from apical and basolateral domains of liver plasma membranes. The caveolae from the two domains were quite homogeneous as studied by immunoaffinity partitioning and electron microscopy. Analysis showed that the caveolae differed in properties in several respects.}},
author = {{Abedinpour, Parisa}},
isbn = {{91-7422-046-2}},
keywords = {{metabolism; Biokemi; Metabolism; Biochemistry; basolateral; apical; immunoaffinity; wheat germ agglutinin; caveolin; caveolae caveolae enriched fraction; plasma membranes; rat lung; rat liver; Affinity Partitioning; two-phase partitioning}},
language = {{eng}},
publisher = {{Biochemistry, Center for Chemistry and Chemical Engineering, Lund University}},
school = {{Lund University}},
title = {{Isolation of caveolae using affinity two-phase partitioning}},
url = {{https://lup.lub.lu.se/search/files/5823347/1693183.pdf}},
year = {{2004}},
}