|Description:||The TR-FRET PPAR alpha Competitive Binding Assay provides a sensitive and robust method for high-throughput screening (HTS) of ligands for peroxisome proliferator-activated receptor alpha (PPAR alpha). The kit uses a terbium-labeled anti-GST antibody, a fluorescent small-molecule pan-PPAR ligand (Pan-PPAR Green), and a human PPAR alpha ligand-binding domain (LBD) that is tagged with glutathione S-transferase (GST), in a homogeneous mix-and-read assay format.
When running the TR-FRET PPAR alpha Competitive Binding Assay, Pan-PPAR Green is added to ligand test compounds followed by addition of a mixture of the PPAR alpha-LBD and terbium anti-GST antibody. When the Pan-PPAR Green is bound to PPAR alpha, energy transfer from the terbium-labeled antibody to the tracer occurs, and a high TR-FRET ratio is observed. Competitive ligand binding to PPAR alpha is detected by a test compound’s ability to displace the tracer, which results in a loss of FRET between the antibody and the tracer. After an incubation period at room temperature, the 520 nm/495 nm TR-FRET ratio is calculated and can be used to determine the IC50 from a dose response curve of the compound. This type of binding assay is analogous to radioligandbased assays, except that it eliminates handling of radioactivity and enables a homogeneous "addition-only" format.
|Applications:||Nuclear Receptor Assay|
|Storage:||Tb-anti-GST Antibody (Rabbit): Store at -20 °C
PPAR alpha-LBD, GST: Store at -80 °C
Fluormone Pan-PPAR Green: Store at -20 °C
TR-FRET PPAR Assay Buffer: Store at 4 °C
1M DTT: Store at -20 °C or -80 °C
|Size:||400 x 40 µL assays|
|Materials Required but Not Supplied:||Microplate Reader|