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Zina Fredj - One of the best experts on this subject based on the ideXlab platform.

Bengt Jergil - One of the best experts on this subject based on the ideXlab platform.

  • purification of caveolae by affinity two phase partitioning using biotinylated antibodies and NeutrAvidin dextran
    Analytical Biochemistry, 2004
    Co-Authors: Irene Barinagarementeria Ramirez, Parisa Abedinpour, Bengt Jergil
    Abstract:

    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. (C) 2004 Elsevier Inc. All rights reserved. (Less)

  • affinity partitioning for membrane purification exploiting the biotin NeutrAvidin interaction model study of mixed liposomes and membranes
    Journal of Chromatography A, 2002
    Co-Authors: Irene Barinagarementeria Ramirez, Sofia Mebrahtu, Bengt Jergil
    Abstract:

    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.

  • affinity partitioning of biotinylated mixed liposomes effect of charge on biotin NeutrAvidin interaction
    Journal of Chromatography B: Biomedical Sciences and Applications, 2000
    Co-Authors: Irene Barinagarementeria Ramirez, Lars Ekblad, Bengt Jergil
    Abstract:

    The partitioning behaviour of biotinylated mixed liposomes in aqueous poly(ethylene glycol)/dextran two-phase systems containing NeutrAvidin-dextran suggests that the biotin-NeutrAvidin affinity interaction is charge dependent. Biotinylated phosphatidylcholine liposomes with a low negative surface charge distributed in the NeutrAvidin-containing bottom phase at neutral pH, but the introduction of additional negative charges by including phosphatidylserine or the surfactant sodium dodecylsulfate in the liposomes caused them to distribute in the poly(ethylene glycol)-rich top phase instead. By gradually lowering the pH of the affinity two-phase system below the isoelectric point (6.3) of NeutrAvidin, negatively charged phosphatidylserine/phosphatidylcholine liposomes increasingly were attracted by NeutrAvidin to the bottom phase. It is suggested that acidic amino acids present at the rim of the biotin-binding pocket of NeutrAvidin may interact electrostatically with charged residues of the closely apposed liposome surface affecting the affinity interaction.

Wing Cheung Mak - One of the best experts on this subject based on the ideXlab platform.

Sawsen Azzouzi - One of the best experts on this subject based on the ideXlab platform.

Laurence Meagher - One of the best experts on this subject based on the ideXlab platform.

  • immobilization and surface characterization of NeutrAvidin biotin binding protein on different hydrogel interlayers
    Journal of Colloid and Interface Science, 2003
    Co-Authors: Patrick Vermette, Thomas R Gengenbach, Upulie Divisekera, Peter Kambouris, Hans J Griesser, Laurence Meagher
    Abstract:

    For a number of potential applications, it is desirable to immobilize avidin class molecules onto solid supports and exploit their ability to bind biotinylated molecules with high affinity. NeutrAvidin molecules were surface immobilized in various ways. In this study, NeutrAvidin was covalently attached by carbodiimide chemistry onto carboxyl groups of polyacrylic acid and carboxymethyl-dextran hydrogel interlayers. A third strategy involved the affinity "docking" of NeutrAvidin onto a biotinylated poly(ethylene glycol) interlayer. These three interlayers were selected for their low nonspecific binding of proteins, which was expected to minimize surface binding of NeutrAvidin by nonspecific interfacial adsorption. X-ray photoelectron spectroscopy (XPS) analyses allowed detailed characterization of the multilayer fabrication steps. An ELISA assay was used to measure NeutrAvidin activity, which varied with the surface immobilization route. Atomic force microcopy (AFM) force measurements showed that the hydrogel interlayer contributed to a repulsive force and verified the specific interaction between biotinylated AFM tips and the NeutrAvidin surfaces. When a solution of free biotin was injected into the AFM liquid cell, the force curve changed substantially and became identical to that recorded between surfaces carrying no NeutrAvidin, indicating that the free solution biotin had displaced NeutrAvidin proteins off the PEG-biotin layer.

  • control over pegylated liposome aggregation by NeutrAvidin biotin interactions investigated by photon correlation spectroscopy
    Langmuir, 2002
    Co-Authors: Patrick Vermette, Sarah Taylor, Dave E Dunstan, Laurence Meagher
    Abstract:

    Photon correlation spectroscopy has been used to determine the degree and sizes of PEGylated-liposome aggregation. The aggregation of the liposomes has been induced by NeutrAvidin−biotin adsorbed to the surfaces of the vesicles. None of the liposome preparations described herein exhibited aggregation before the introduction of NeutrAvidin. The critical ratios of the NeutrAvidin−biotin required to induce vesicle aggregation have been measured and are reported in this work. The aggregation is found to be dependent upon the ratio of NeutrAvidin to exposed biotinylated lipid and also the exposed biotinylated lipid fraction.