Zusammenfassung
The detection of protein-protein binding on microarrays using the fluorescence lifetime as a dynamic analytical parameter was investigated in a model system. The assay is based on Forster resonance energy transfer (FRET) and carried outwith biotinylated Bovine Serum Albumin and streptavidin, labeled with the commonly used microarray dyes Alexa 555 and Alexa 647, respectively. This efficient FRET ...
Zusammenfassung
The detection of protein-protein binding on microarrays using the fluorescence lifetime as a dynamic analytical parameter was investigated in a model system. The assay is based on Forster resonance energy transfer (FRET) and carried outwith biotinylated Bovine Serum Albumin and streptavidin, labeled with the commonly used microarray dyes Alexa 555 and Alexa 647, respectively. This efficient FRET donor/acceptor pair was employed in a competitive assay format on three different microarray surfaces. The fluorescence was excited by 200 ps laser pulses from a mode-locked and cavity-dumped argon-ion laser, adapted to an intensified CCD camera as detection unit allowing time resolution with subnanosecond precision. Lifetime maps were recorded according to the Rapid Lifetime Determination (RLD) scheme. Interaction between the proteins could clearly be detected on all formats and resulted in almost complete quenching on CEL Epoxy surfaces upon addition of excess streptavidin labeled the FRET acceptor dye. In this case, the fluorescence lifetimes dropped by 90%, whereas on ARChip Epoxy and ARChip Gel the reduction was 54% and 47%, respectively. Good linearity of the quenching curve was obtained in all cases. The method is applicable to all types of protein interaction analysis on microarrays, particularly in cases where evaluation of fluorescence intensity is prone to erroneous results and a more robust parameter is required. (C) 2008 Elsevier B.V. All rights reserved.
Altmetric