Homogeneous GTP Binding Assay Employing QRET Technology

Quenching resonance energy transfer (QRET): a single-label technique for inhibitor screening and interaction studies

“…The increased distance decreases energy transfer between the Ln 3+ -ligand and the quencher, thus increasing the luminescence lifetime of the Ln 3+ -chelate and the monitored overall TRL-signal in the measurement window from 400 to 800 ms [9]. The labeled ligand could be, for example, a DNAfragment, peptide, nucleotide or small hormone incapable of protecting the label as such [8][9][10][11]. The QRET based screening is easy to convert to new targets if a small molecule ligand of the target can be labeled.…”

A Homogeneous Single-Label Time-Resolved Fluorescence cAMP Assay

“…dIScuSSIon previously, the potential of the Qret technique has been demonstrated for receptor-ligand and Gtp binding studies on cells and cell membranes. 11,13 Here we have developed a cellbased homogeneous assay for the second messenger, cAMp, using a time-resolved fluorescence single-label approach. the cAMp assay was developed using a stably transfected HeK293 i cell line overexpressing human β 2 Ars.…”

“…We have previously demonstrated that receptor-ligand and Gtp binding assays can be constructed on the Qret concept. 11,13 Fluorescence polarization is another known single-label approach for drug-screening purposes. However, the drawback of this method is the use of conventional fluorescence having 3-4 orders of magnitude less sensitive detection limit than timeresolved fluorescence 24 and a low s/B ratio, 4,12,25,26 reducing the applicability of the method for many target molecules.…”

“…the assay is based on the quenching resonance energy transfer (Qret) technique [11][12][13] using a single-label approach and soluble quenchers affecting the fluorescence of the unbound labeled binding component. less fluorescence quenching is observed when the labeled component binds to an antibody.…”

A Homogeneous Method to Measure Nucleotide Exchange by α-Subunits of Heterotrimeric G-Proteins Using Fluorescence Polarization

“…14 The ability to use QRET in a homogenous format (i.e., without the need for separation of bound and unbound Eu-GTPgS) represents an advance over the earlier TR-FRET-based assays. 15 Additional, non-lanthanide-based fluorescent GTP analogs have also led to the establishment of nonradioactive assays to quantify the nucleotide cycling properties of G-proteins [16][17][18] ; however, these alternate assays involve monitoring changes in the absolute intensity of the fluor as its local solvating environment changes upon binding or hydrolysis events. The need to measure absolute intensity change, coupled with the use of fluors in the green range, prevents these assays from being suitable for screening small molecule libraries for nucleotide-state modulators.…”