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The GeneBLAzer® PPAR alpha-UAS-bla HEK 293T Cellular Assay provides an accurate, sensitive, and easy-to-use method for monitoring the cellular response of PPAR alpha to drug candidates or other stimuli.
Features of the The GeneBLAzer® PPAR alpha-UAS-bla HEK 293T Cellular Assay: • Convenience—plate cells and perform assay without additional cell culture steps • Low Noise—the kit uses a Fluorescence Resonance Energy Transfer (FRET)-based ratiometric readout for minimum noise and error • Low Background—the GAL4 fusion prevents any background from endogenous receptor
The Cells The GeneBLAzer® PPAR alpha-UAS-bla HEK 293T cells contain the ligand-binding domain (LBD) of the human peroxisome proliferator-activated receptor alpha (PPAR alpha) fused to the DNA-binding domain of GAL4 transiently transduced (via BacMam virus) into the GeneBLAzer® UAS-bla HEK293T cell line. GeneBLAzer® UAS-bla HEK 293T cells stably express a β-lactamase reporter gene under the transcriptional control of an upstream activator sequence (UAS).
The Assay When an agonist binds to the LBD of the GAL4 (DBD)-PPAR delta (LBD) fusion protein, the protein binds to the UAS, resulting in expression of β-lactamase. The FRET ratiometric readout from the live-cell substrate reduces the absolute and relative errors that can mask the underlying biological response of interest. PPAR alpha UAS-bla HEK 293T cells have been tested for assay performance using variable assay conditions, including cell number, stimulation time, substrate loading time and have been validated for Z′ and EC50 concentrations of GW7647. Additional testing data using alternate stimuli are also available.
Speed and Convenience Because PPAR alpha UAS-bla HEK 293T cells are transiently transduced with BacMam virus, they are ready to be used in an assay without any additional cell culture steps. Just plate the cells and assay as outlined in the protocol.
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