Affinity analysis of a protein-aptamer complex using nonequilibrium capillary electrophoresis of equilibrium mixtures.
We propose a new method that allows the use of low-affinity aptamers as affinity probes in quantitative analyses of proteins. The method is based on nonequilibrium capillary electrophoresis of the equilibrium mixture (NECEEM) of a protein with its fluorescently labeled aptamer. In general, NECEEM of a protein with a fluorescently labeled aptamer generates an electropherogram with three characteristic features: two peaks and an exponential curve. Two peaks correspond to (i) the equilibrium amount of free aptamer in the equilibrium mixture and (ii) the amount of the protein-aptamer complex that remains intact at the time of detection. The exponential part is ascribed to the complex decaying during separation under nonequilibrium conditions. Simple analysis of the three features in experiments with known concentrations of the protein can be used for the determination of the equilibrium dissociation constant, Kd, of the aptamer-protein complex. Similar analysis of the three features in the experiment with unknown concentration of the protein and known Kd value allows the determination of the protein concentration. In this proof-of-principle work, the NECEEM method was applied to the analysis of thrombin using a fluorescein-labeled aptamer under the conditions at which the protein-aptamer complex completely decayed during the separation. We demonstrated that, despite the decay, as few as 4 x 10(6) molecules of the protein could be detected with NECEEM without sacrificing the accuracy. This sensitivity is comparable with that reported by others for the aptamer-based equilibrium method. Thus, the proposed NECEEM-based method allows the use of aptamers for highly sensitive affinity analysis of proteins even when protein-aptamer complexes are unstable.
Berezovski
Maxim
M
Department of Chemistry, York University, Toronto, Ontario, Canada M3J 1P3.
Nutiu
Razvan
R
Li
Yingfu
Y
Krylov
Sergey N
SN
D000345:Affinity Labels; D019075:Electrophoresis, Capillary / Q000379:methods*; D005456:Fluorescent Dyes; D006801:Humans; D011506:Proteins / Q000032:analysis*; D013917:Thrombin / Q000032:analysis
D000345:Affinity Labels; D005456:Fluorescent Dyes; D011506:Proteins; D013917:Thrombin