Flow-through partial-filling affinity capillary electrophoresis can estimate binding constants of neutral ligands to receptors via a competitive assay technique
✍ Scribed by John Kaddis; Erica Mito; Joseph Heintz; Alfredo Plazas; Frank A. Gomez
- Publisher
- John Wiley and Sons
- Year
- 2003
- Tongue
- English
- Weight
- 109 KB
- Volume
- 24
- Category
- Article
- ISSN
- 0173-0835
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✦ Synopsis
This work evaluates the use of a competitive binding assay using flow-through partial-filling affinity capillary electrophoresis (FTPFACE) to estimate binding constants of neutral ligands to a receptor. We demonstrate this technique using, as a model system, carbonic anhydrase B (CAB, EC 4.2.1.1) and arylsulfonamides. In this technique, the capillary is first partially filled with a negatively charged ligand, a sample containing CAB and two noninteracting standards, and a neutral ligand, then electrophoresed. Upon application of a voltage the sample plug migrates into the plug of negatively charged ligand (L(-)) resulting in the formation of a CAB-L(-) complex. Continued electrophoresis results in mixing between the neutral ligand (L(0)) and the CAB-L(-) complex. L(0) successfully competes out L(-) to form the new CAB-L(0) complex. Analysis of the change in the relative migration time ratio (RMTR) of CAB relative to the noninteracting standards, as a function of neutral ligand concentration, yields a value for the binding constant. These values are in agreement with those estimated using other binding and ACE techniques. Data demonstrating the quantitative potential of this method is presented.