GeneBLAzer beta-lactamase Reporter Technology provides you with the most reliable, integrated set of tools for accelerating your cell-based target validation, pathway analysis, and compound screening in drug discovery.
This proven technology combines molecular and cell biology and a Fluorescence Resonance Energy Transfer (FRET)-based, ratiometric detection method that reduces experimental noise which can lead to false hits.
The GeneBLAzer Technology consists of readily accessible tools and custom assays (click table below) for studying numerous target classes and cellular processes, including: surface and intracellular reporters (such as nuclear and cytokine receptors, orphan and known G-protein coupled receptors), a wide range of signal transduction pathways, ion channels, other transporters, and transcriptional regulators (including kinases and intracellular processes).
Integrated Solutions Using GeneBLAzer Reporter Technology
GeneBLAzer Technology uses a mammalian-optimized bla gene combined with a FRET-enabled substrate to provide reliable and sensitive detection in cells.
Cells are loaded with an engineered fluorescent substrate containing two fluoroprobes, coumarin and fluorescein. In the absence of bla expression, the substrate molecule remains intact. In this state, excitation of the coumarin results in fluorescence resonance energy transfer to the fluorescein moitey and emission of green light. However, in the presence of bla expression, the substrate is cleaved, separating the fluorophores, and disrupting energy transfer. Excitation of the coumarin in the presence of enzyme bla activity results in a blue fluorescence signal. The resulting blue:green ratio provides a normalized reporter response.
To compare the effects of some types of experimental noise on ratiometric and non-ratiometric reporter readouts, data were generated using assay conditions that included variations in cell number and substrate concentration. Cells constitutively expressing beta-lactamase were plated in varying degrees of confluency and LiveBLAzer-FRET B/G Substrate was added in either the recommended (1X) or high (4X) concentration.
HEK 293T cells expressing beta-lactamase coupled to a cyclic AMP response element (CRE) were stably transfected with a cDNA encoding a G-Protein Coupled Receptor. Cells were then treated with increasing concentrations of several agonists and analyzed using LiveBlazer-FRET B/G Substrate. The data shows the ability of the GeneBLAzer Technology to establish rank order of potency and to distinguish types of agonism, even when the differences between them are small. Dose response curves shown are full agonists (Compounds 1 and 2), partial agonists (Compounds 3 and 4) and an inverse agonist (Compound 5).
5HT1a-G_NFAT-bla CHO-K1 cells were run in agonist mode with the LOPAC 12080TM library of small molecules using Fluo-4 multiplexed with the LiveBLAzer-FRET B/G Assay. Cells were loaded with Fluo-4 to search for compounds that trigger the Ca2+ flux upon binding to the serotonin receptor. The same cells were loaded with LiveBLAzer-FRET B/G substrate and then analyzed. Hits registered with Fluo-4 are shown in Panel 1, and hits registered with LiveBLAzer-FRET B/G are shown in Panel 2.
Out of 18 known serotonin receptor agonists detected by the LiveBLAzer Assay, 16 were also detected by the Fluo-4 Assay. In contrast, 4 other compounds detected by LiveBLAzer Assay were not detected by the Fluo-4 Assay, while 6 additional compounds detected by the Fluo-4 Assay were not detected by the LiveBLAzer Assay. By multiplexing these two assay technologies, one can validate true hits, while eliminating those that are not.
For Research Use Only. Not for use in diagnostic procedures.