GeneBLAzer® Technology Overview
How GeneBLAzer® Technology works
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.
Ratiometric Advantage of GeneBLAzer® Technology: Minimize the Effects of Experimental Noise
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.
Data for the ten samples was then analyzed using either the blue/green (ratiometric) or blue (non-ratiometric) values from this assay. The data illustrates the ratiometric readout reduces both absolute and relative errors caused by experimental noise compared to non-ratiometric methods.
Full, Partial, and Inverse Agonists Distinguished using GeneBLAzer® Technology
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).
Multiplexing GeneBLAzer® Technology with Fluo-4
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.