A flow injection kinase assay system based on time-resolved fluorescence resonance energy-transfer detection in the millisecond range.
A flow injection analysis (FIA) system for biochemical assays using time-resolved fluorescence resonance energy transfer (TR-FRET) in the millisecond time scale was developed. As a model system, we studied a kinase assay, measuring the phosphorylation of poly(GT)-biotin (substrate) by a receptor tyrosine kinase (epidermal growth factor receptor). A streptavidin labeled with XL665 (SA-XL665)-the acceptor-was coupled to the biotin moiety, and an antiphosphotyrosine antibody labeled with europium cryptate (Ab-EuK)-the donor-was coupled to the phosphorylated tyrosine group(s). Long-lived FRET can only occur if the substrate is successfully phosphorylated. For the time-resolved detection of such long-lived luminescence phenomena in a flow system, the repetition rate of the excitation source plays a crucial role. Good results were obtained for a small-sized commercially available quadrupled Nd:YAG laser emitting at 266 nm with a repetition rate of 7.8 kHz and a pulse width of 0.3 ns. The long-lived emissions of the donor at 625 nm and that of the acceptor at 665 nm were monitored simultaneously with two photomultipliers, using a delay time of 50 micros and a gate time of 75 micros to exclude background fluorescence interferences. In the FIA experiments, the Ab-EuK concentration was 6 nM and the substrate concentration and SA-XL665 concentrations were 7 nM. By monitoring the intensity changes at 625 and 665 nm, the inhibition of tyrosine kinase by tyrphostin AG1478 was studied and an IC(50) value of 5.1 +/- 0.4 nM obtained.
Laser Center, Department of Analytical Chemistry and Applied Spectroscopy, Vrije Universiteit Amsterdam, de Boelelaan 1083, 1081 HV Amsterdam, The Netherlands.
Junko
J
Hirata
Camiel F
CF
de Jong
Maarten M
MM
van Dongen
Joost
J
Buijs
Freek
F
Ariese
Hubertus
H
Irth
Cees
C
Gooijer
D001665:Binding Sites; D066246:ErbB Receptors / Q000032:analysis* / Q000378:metabolism; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D006801:Humans; D007700:Kinetics; D013997:Time Factors
D066246:ErbB Receptors