By J. J. Duistermaat, J. A. C. Kolk, J. P. van Braam Houckgeest
1. Differentiation -- 2. Integration
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Additional resources for Multidimensional real analysis. 1 Differentiation
22 Ilm filter and degassed before use. 4 Regeneration Solution For repeated measurements on the same sensor chip, the surface may be regenerated by removal of analyte and other non-covalently bound material. Commonly used solutions for regeneration include high salt concentrations and/or high or low pH. Regenerating conditions should be chosen to ensure complete elution of the associated analyte without denaturing or inactivating the immobilized ligand. 3 Immobilizing Ligand Methods for attaching the ligand to the sensor surface may be divided into two classes: direct immobilization, where the ligand molecules are covalently linked to the dextran layer on the sensor surface, and capturing, where the ligand binds with a high affinity to immobilized capturing molecules.
In experiments for qualitative determination of whether the analyte binds or not, careful adjustment of the buffer is not critical. However, for quantitative kinetic determinations using a series of different analyte concentrations, it is important that buffer exchange is performed reliably, for instance by dialysis or gel filtration. Bulk effects can be subtracted at the data analysis stage if a blank control is included in the experiment. This will also correct for non-specific adsorption of the sample on the surface of the sensor chip.
Lecular interactions, but low molecular weight analytes often exhibit fast association and dissociation and reach steady state in a short time. The flow rate may be kept low (about 5 Ill/min) to minimize sample consumption. Generally speaking, the larger the dissociation rate constant (kd)' the shorter the time needed to reach equilibrium and vice versa. The values of Req obtained for various concentrations of analyte are plotted against the concentration of analyte, and the dissociation constant (KD ) is derived by non-linear curve fitting.