Affinity Two-Phase Partitioning of Liposomes and Membranes

Abstract: Affinity two-phase partitioning based on an immunoaffinity sandwich approach for the rapid and selective purification of membranes is presented in this thesis. The method was developed by studying different parameters governing the affinity partitioning of model membranes. To this end, biotinylated liposomes were used. They specifically distributed to the bottom phase of a poly (ethylene glycol)/dextran two-phase system through interactions with the affinity ligand, NeutrAvidin coupled to dextran. Restrictions of the liposomal biotin-NeutrAvidin affinity interaction in the two-phase system were analysed. The immunoaffinity sandwich approach exploits the very strong interaction between NeutrAvidin and biotin and the introduction of specific antibodies (a primary and a biotinylated secondary antibody) makes the method highly selective. As an example, caveolae from different sources were purified by affinity-two-phase partitioning, yielding a material of the same or better purity as when purified by standard procedures. The same approach, employing other selective primary antibodies, should facilitate the purification of other membrane fractions. The use of biotinylated secondary antibodies in the immunoaffinity sandwich approach obviates the need of biotinylating each primary antibody for each application, facilitating the general applicability of the method. A miniaturised version of affinity two-phase partitioning in levitated drops (< 1 µl) was investigated using biotinylated liposomes as model material and NeutrAvidin-dextran as affinity ligand. Several factors affecting the affinity partitioning in a miniature system, including control of evaporation, and addition of minute amounts of material to the drop via special dispensers, were investigated. Phase separation was followed visually by microscopy and phase extraction was performed by specifically made micropipettes. Biotinylated liposomes were partitioned to the PEG-rich phase in the absence of the NeutrAvidin ligand and to the dextran-rich phase in its presence, similarly to their partition in larger systems. This miniaturised technique would allow the separation of membranes from single cells for analysis as well as being suitable for screening of separation conditions.