Biacore SPR Lab Report

Figure 1: Schematic illustration of the principle of Biacore SPR to measure the interaction between analyte molecules in the flow with immobilized receptor binding sites on the surface. A) SPR uses an optical method to measure changes in the refractive index near the surface of a thin gold film on a glass slide. At a critical angle of incident light on the surface-solution interface an electromagnetic field or evanescent wave is generated. This is detected as absorption of light and a decrease in the intensity of the reflected light. The generated evanescent wave extends a short distance (≈ 300 nm) into the solution and is sensitive to changes in the medium. When molecules in the analyte flow binds to receptor binding sites at the surface the
To overcome this, a very common strategy is to use secondary molecules such as antibodies, to capture the target protein on the surface. An antibody that recognizes the membrane protein of interest can be immobilized covalently on the surface and subsequently capture the target protein when it is injected. Opposite to covalent immobilization, where proteins are immobilized randomly, the availability of monoclonal antibodies directed against specific epitopes on the target protein makes it possible to immobilize proteins in controlled orientations
In addition to stabilize the biological function of captured membrane proteins, nanodiscs have some benefits when used together with SPR. Nanodiscs provide access to both sides of the lipid bilayers. Thereby it is possible to avoid problems with orientation and access to integral membrane proteins that can occur when lipid vesicles or supported membrane mono- or bilayers are used to capture membrane proteins on the sensor surface. In addition, the MSP encircling the nanodisc lipidbilayer can increase the flexibility of immobilization strategies; the MSP can be genetically engineered with various affinity tags useful for immobilization on a complementary antibody sensor surface. To date, the MSP has been generated with his- and Flag-tags and recently a C9 epitope