Biomedical subjects

Bengt Jergil

Publications and source records attributed to Bengt Jergil.

4 recordsLinked to original sources

PubMed2004

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.

Animals↗

PubMed2004

Affinity two-phase partitioning in acoustically levitated drops.

Miniaturized (<1 microL) biospecific affinity two-phase partitioning in an acoustically levitated drop is described. Miniaturization commonly gives unfavorable surface/volume ratios, but in the levitation approach adsorption problems are minimized since the only surrounding wall is the liquid/air interface of the drop. Biotinylated liposomes were partitioned in aqueous poly(ethylene glycol)/dextran two-phase drops with NeutrAvidin-dextran as the affinity ligand. A two-phase drop was trapped and manipulated in a node of a standing ultrasonic wave. Alternatively, a two-phase system was formed by levitation and evaporation of a polymer one-phase drop. Phase mixing was achieved by adjusting the ultrasonic field and phase separation by readjusting the field. NeutrAvidin-dextran brought about the redistribution of biotinylated liposomes from the poly(ethylene glycol)-rich phase into the dextran-rich phase. Thus, an entire affinity two-phase separation procedure, including mixing of the phases and incubation to allow affinity interactions to develop under constant volume, followed by phase separation under controlled evaporation, can be performed in a single levitated drop. This miniaturized technique would allow the separation of biologically active membranes or organelles from individual cells for analysis.

Journal Article↗

PubMed2003

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

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.

Actins↗

PubMed2002

Affinity partitioning for membrane purification exploiting the biotin-NeutrAvidin interaction. Model study of mixed liposomes and membranes.

Biotinylated negatively charged liposomes as well as membranes were affinity partitioned in an aqueous poly(ethylene glycol)-dextran two-phase system using NeutrAvidin conjugated to dextran as affinity ligand. Both liposomes and membranes redistributed from top to bottom phase upon addition of NeutrAvidin-dextran. The presence of 35-60 mM Li2SO4 was necessary both to force the components into the top phase without ligand and for ligand-dependent redistribution into the bottom phase. Attaching biotin via a hexanamidohexanoyl spacer and an increased density of biotin or NeutrAvidin enhanced the affinity separation. The separation conditions in these model experiments provide a basis for affinity partitioning of membranes using other affinity ligands.

Avidin↗