Depending on the application a known amount of ligand must be immobilized on the sensor chip surface. A crude guideline is the figure about immobilization levels.
In general, for kinetic measurements, a total analyte response of maximal 100 RU, when the analyte is injected (1), (2) is desired. With this value in mind (R max), the amount of ligand (in response units) to be immobilized can be calculated with:
Or more conveniently:
This formula is theoretical because it assumes that all immobilized ligand molecules are fully accessible and functional, which is most likely not the case with the standard amine coupling. When the Rmax is known after analyte injection, the percentage of functional ligand (the ligand that can bind the analyte) can be calculated. A low analyte response can be caused by low affinity, an impure ligand or that binding sites have been affected by the immobilization procedure. A to high response can be induced by non-specific binding or analyte aggregates (6).
Because the dextran matrix has a significant extension (in of the order of 100 nm for sensor chip CM5), "surface concentrations" on the sensor chip surface are strictly speaking volume concentrations. The relation between the actual protein 'concentration' and the Response units (RU) for the Biacore type machines (7), (8) is:
These relations make it possible to calculate the ligand concentration:
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|(5)||BIACORE AB BIACORE Technology Handbook. (1998).|
|(6)||BIACORE AB BIA Symposium '98. (1998).|
|(7)||BIACORE AB BIACORE Getting Started. (1998).|
|(8)||Muller, K. M. et al Model and simulation of multivalent binding to fixed ligands. Analytical Biochemistry 261: 149-158; (1998).|