Hydrophobic capture

Membrane-associated proteins present special problems for study in aqueous environments, particularly in cases where water-soluble fragments do not adequately mimic the function of the intact protein. Two essentially distinct approaches, based respectively on lipid monolayers and bilayers, are available for retaining a hydrophobic environment for ligands attached to the sensor chip surfaces.

Bilayer attachment

Several approaches are available for attachment of intact membrane structures with associated ligands to the sensor surface. Proteins incorporated into liposomes may be immobilized on the sensor chip surface. Liposomes may be prepared with a specific antigenic component or with biotinylated lipids, allowing capture of the liposomes with immobilized antibody or streptavidin respectively. Alternatively, Sensor Chip L1 may be used: this surface carries hydrophobic structures on the dextran matrix that can insert into liposomes and serve to attach the membranes to the dextran matrix.

Liposomes attach to the hydrophobic anchors in the dextran matrix on Sensor Chip L1 and can fuse to form a supported lipid bilayer.

A variant of this approach, called on-surface reconstitution (OSR), involves immobilization of detergent-solubilized transmembrane protein on the carboxymethylated dextran matrix of Sensor Chip L1, followed immediately by replacement of the detergent by lipid to reconstitute membrane structures around the immobilized protein. Under suitable conditions, this will result in formation of a supported lipid bilayer separated from the surface of the sensor chip by an aqueous region.

On surface reconstitution of supported lipid bilayers with integrated membrane proteins.(this is a simplified representation).

Monolayer attachment

Sensor Chip HPA lacks a dextran matrix, so that the hydrophobic alkanethiol layer covering the gold film is directly exposed to the solution. Adsorption of lipids from micelles or liposomes to this surface creates a lipid monolayer with the hydrophobic lipid tails oriented towards the gold film and the hydrophilic heads towards the aqueous sample. This structure can support interaction studies with membrane-associated ligands that are introduced into the lipid preparation before adsorption to the surface.

Adsorption of liposomes on Sensor Chip HPA results in the formation of a lipid monolayer covering the chip surface.

While this approach provides a stable environment for ligands associated with a membrane surface or partially inserted into the membrane, it is not suitable for more deeply inserted or trans-membrane proteins since the surface represents only half the membrane structure. Lipid bilayers can be supported in various ways on the surface for studies that require complete membrane structures.

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