Sensor chip HPA surface is composed of long chain octadekanethiol molecules that form a flat, quasi-crystalline hydrophobic layer. It is designed to facilitate liposome mediated hydrophobic adsorption of a user-defined polar lipid monolayer. In addition to the components essential for the formation of the liposome, other membrane bound molecules can be incorporated. These molecules remain embedded in the lipid monolayer after the liposome mediated hydrophobic adsorption process is completed and give a particular binding specificity to the surface. A complete coverage of the surface with a polar lipid monolayer will practically eliminate non-specific hydrophobic binding of proteins and provide a surface suitable for interaction studies.
The recommended temperature for analysis is 25°C or below. A hydrophobic surface has an inherent affinity for the microscopic air-bubbles that tend to form in aqueous solutions at elevated temperatures. Therefore, the buffers must be degassed. If experiments are performed above 25°C or at low flow rates, degassing must be very thorough to secure undisturbed data recording. The eluent buffer should always be detergent-free, freshly prepared, filtered through a 0.22 µm and degassed.
The adsorption of lipids starts with a wash of the surface with a five-minute injection of 40 mM octyl-glucoside (n-Octyl Β-D-Glucopyranoside) in water at a flow rate of 5 - 10 µl/min and ends with an extraclean command.
The injection of the liposomes sample should start directly after the wash with octyl-glucoside. The liposomes are preferably prepared in a buffer identical with the eluent buffer. A concentration of 0.5 mM with respect to phospholipids is commonly sufficient (1). The process of coating Sensor chip HPA with a lipid monolayer typically takes 0.5-3 hours depending on the temperature, concentration (2), type of liposome, and eluent buffer. Sometimes the coating procedure can be completed in minutes whereas other liposome preparations may require an overnight procedure for optimal coating. Small liposomes, obtained e.g. by extrusion with a 50 nm pore size filter, will diffuse faster and are more prone to fuse with the surface than liposomes with a large diameter, thereby shortening the adsorption process. Another parameter of relevance for the adsorption is the phase transition temperature (Tc), which is related to the liposome composition. Typically, a flow rate of 2 - 10 µl/min is used for liposome adsorption. The process of spontaneous fusion of liposomes is monitored until the sensorgram readings begin to level out. This will often occur at the response level that is in the order of 2000 - 4000 RU or more above the original baseline.
Membrane associated proteins are best deposited on the chip together with the lipid vesicles (3).
At this stage, the surface may carry multiple lipid layers and partially fused liposomes. Such loosely bound structures can be washed away by briefly increasing the flow rate to 100 ml/min. Alkaline pH will often produce an additional decrease in the sensorgram reading, down to a level which will remain as a stable baseline. If compatible with the user-defined reagents one short pulse of 10-100 mM NaOH can be used for this purpose (3),(1).
The hydrophobic surface on Sensor chip HPA has a strong tendency to bind proteins non-specifically. This tendency is reduced or eliminated when the HPA surface is coated with a lipid monolayer. An application-specific negative control should be included in the investigation to ensure that the observed binding is relevant. A standard protein such as BSA may also be used to assess the extent of coverage of the surface. Using a 5-minute injection of BSA at 0.1 mg/ml in eluent buffer, an uncoated, Octyl glycoside washed surface will bind about 1000 RU, whereas a surface fully coated with DMPC (dimyristoyl phosphatidylcholine) or POPC (palmitoyleoyl phosphatidylcholine) typically binds less than 100 RU.
With the lipid monolayer covering the surface, subsequent experimental strategies and data interpretation will be essentially the same as for experiments using Sensor chip CM5, an exception being the requirement for detergent-free buffers.
Analytes that have bound to the lipid monolayer or to ligands anchored in the monolayer may be dissociated with variations in e.g. pH or ionic strength, leaving the lipid monolayer intact for additional cycles of interaction. Lipid monolayers composed of e.g. DMPC or POPC are quite robust in this respect and can withstand exposure to 100 mM NaOH and 100 mM HCl. However, user-defined binding sites may be the limiting factor for what agents can be used.
Detergents and organic solvents will alter or destabilize the lipid monolayer or even cause it to disintegrate. Although such agents can be used in attempts to strip the lipid monolayer from the surface in preparation for adsorbing a new monolayer, biacore does not guarantee the performance specifications of such regenerated surface.
|(1)||Webster, C. I. et al Kinetic analysis of high-mobility-group proteins HMG-1 and HMG-I/Y binding to cholesterol-tagged DNA on a supported lipid monolayer. Nucleic Acids Res. 28: 1618-1624; (2000).|
|(2)||Hubbard, J. B. et al Self assembly driven by hydrophobic interactions at alkanethiol monolayers: mechanisms of formation of hybrid bilayer membranes. Biophysical Chemistry 75: 163-176; (1998).|
|(3)||Cooper, M. A. et al Surface plasmon resonance analysis at a supported lipid monolayer. Biochim.Biophys.Acta 1373: 101-111; (1998).|