Protein-aptamer binding studies using microchip affinity capillary electrophoresis.
The use of traditional CE to detect weak binding complexes is problematic due to the fast-off rate resulting in the dissociation of the complex during the separation process. Additionally, proteins involved in binding interactions often nonspecifically stick to the bare-silica capillary walls, which further complicates the binding analysis. Microchip CE allows flexibly positioning the detector along the separation channel and conveniently adjusting the separation length. A short separation length plus a high electric field enables rapid separations thus reducing both the dissociation of the complex and the amount of protein loss due to nonspecific adsorption during the separation process. Thrombin and a selective thrombin-binding aptamer were used to demonstrate the capability of microchip CE for the study of relatively weak binding systems that have inherent limitations when using the migration shift method or other CE methods. The rapid separation of the thrombin-aptamer complex from the free aptamer was achieved in less than 10 s on a single-cross glass microchip with a relatively short detection length (1.0 cm) and a high electric field (670 V/cm). The dissociation constant was determined to be 43 nM, consistent with reported results. In addition, aptamer probes were used for the quantitation of standard thrombin samples by constructing a calibration curve, which showed good linearity over two orders of magnitude with an LOD for thrombin of 5 nM at a three-fold S/N.
Gong
Maojun
M
Department of Chemistry, University of Cincinnati, Cincinnati, OH 45221-0172, USA.
Nikcevic
Irena
I
Wehmeyer
Kenneth R
KR
Limbach
Patrick A
PA
Heineman
William R
WR
D000327:Adsorption; D052158:Aptamers, Peptide / Q000378:metabolism*; D046209:Electrophoresis, Microchip / Q000379:methods*; D011485:Protein Binding; D013917:Thrombin / Q000737:chemistry
D052158:Aptamers, Peptide; D013917:Thrombin