Through-a-chip partial filling affinity capillary electrophoresis for estimating binding constants of ligands to receptors.
In this paper, we describe the development of a microfluidic/capillary electrophoresis (CE) technique employing partial filling affinity capillary electrophoresis (PFACE) to estimate binding constants of ligands to receptors using as model systems carbonic anhydrase B (CAB, EC 4.2.1.1) and vancomycin from Streptomyces orientalis. Using multilayer soft lithography (MSL), a microfluidic device (MD) consisting of fluid and control channels is fabricated and fitted with an external capillary column. Multiple flow channels allows for manipulation of a zone of ligand and sample containing receptor and non-interacting standards into the MD and subsequently into the capillary column. Upon electrophoresis the sample components migrate into the zone of ligand where equilibrium is established. Changes in migration time of the receptor are used in the analysis to obtain a value for the binding interaction. The manipulation of small volumes of solution on the MD minimizes the need of time-consuming pipetting steps.
Brown
Abby
A
Department of Chemistry and Biochemistry, California State University, Los Angeles, 5151 State University Drive, Los Angeles, CA 90032-8202, USA.
Morales
Christina
C
Gomez
Frank A
FA
D019075:Electrophoresis, Capillary / Q000379:methods*; D008024:Ligands; D011485:Protein Binding; D011956:Receptors, Cell Surface / Q000378:metabolism*
D008024:Ligands; D011956:Receptors, Cell Surface