Assay Method Information

Assay Name:  SPR Assay
Description:  The anti-His antibody (His Capture Kit, GE Healthcare) was immobilized on sensor chip CM5 (GE Healthcare), in a Biacore T200 or Biacore S200 instrument, to ∼10000 resonance units (RU) using a standard amine coupling procedure (Amine Coupling Kit, GE Healthcare). His-tagged aldose reductase (ALR2), at a concentration of 100 μg/mL, was captured on the anti-His antibody using dual injection consisting of a 3 min injection of protein followed by a 100 s activation with a mixture (1:1) of 0.2 M N-ethyl-N-(dimethylamino)propylcarbodiimide (EDC) and 50 mM N-hydroxysuccinimide (NHS), and final blocking with an injection of 1.0 Methanolamine (pH 8.5) for 100 s. Activation with EDC and NHS and blocking with ethanolamine stabilized the ALR2 capture and allowed the use of a single surface for the analysis of a number of compounds with no need for regeneration. The ALR2 capture level was ∼1000 RU, and all injections were performed at a flow rate of 10 μL/min. For the interaction analysis, the compound concentrations ranged from 3 to 10000 nM with a 5-fold dilution step, using a single-cycle kinetic approach with three or five concentrations or injections per cycle.
Affinity data for this assay
 

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