PMID:18232512
| Citation |
Ramirez, A and Gomez, FA (2007) Use of voltage gradient partial-filling affinity capillary electrophoresis to estimate binding constants of ligands to receptors. J Capill Electrophor Microchip Technol 10:43-50 |
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| Abstract |
Voltage gradient partial-filling affinity capillary electrophoresis (VGPFACE) is used to determine binding constants between carbonic anhydrase B (CAB, E.C.4.2.1.1) and arylsulfonamides, and vancomycin (Van) from Streptomyces orientalis and teicoplanin (Teic) from Actinoplanes teicomyceticus and D-Ala-D-Ala terminus peptides. Two variations of VGPFACE are described herein. In the first technique, the capillary is partially filled with ligand at increasing concentrations followed by a sample containing receptor and two noninteracting standards and electrophoresed in buffer using a voltage gradient that increases from 0 to 25 kV over the duration of the experiment. Upon continued electrophoresis, zones of solution overlap, and equilibrium is established between the ligand and receptor, causing a shift in the migration time of the receptor with respect to the noninteracting standards. This change in migration time is utilized for estimating a binding constant (K(b)). In the second technique, voltage gradient partial-filling multiple-injection ACE (VGPFMIACE), a multiple-injection sequence is used whereby the capillary is partially filled with ligand at increasing concentrations, a noninteracting standard, three or four separate plugs of receptor each separated by small plugs of buffer, and a plug containing a second noninteracting standard; this is then electrophoresed in buffer with a similar voltage gradient. Upon continued electrophoresis, a similar equilibrium is established and a value for K(b) is obtained for the interaction. The VGPFACE technique expands the functionality and potential of ACE as an analytical tool to examine various receptor-ligand interactions. |
| Links | |
| Keywords |
Binding, Competitive; Carbonic Anhydrase I/chemistry; Chromatography, Affinity/methods; Electrophoresis, Capillary/instrumentation; Electrophoresis, Capillary/methods; Glycopeptides/chemistry; Kinetics; Ligands; Motion; Oligopeptides/chemistry; Protein Binding; Reproducibility of Results; Static Electricity |
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