Transcreener® ADP² TR-FRET Assay

Transcreener® HTS is a universal, high throughput biochemical assay platform based on the detection of nucleotides, which are formed by thousands of cellular enzymes - many of which catalyze the covalent regulatory reactions that are central to cell signaling and are of great value as targets in drug discovery.

The Transcreener® TR-FRET Assays are a single step, competitive immunoassay for direct detection of nucleotides with a far red time-resolved Förster-resonance-energy-transfer (TR-FRET) readout. The reagents for all of the assays are a far red Tracer bound to a highly-specific monoclonal antibody-Terbium conjugate. Excitation of the Terbium complex in the UV range (ca. 330 nm) results in energy transfer to the Tracer and emission at a higher wavelength (665 nm) after a time delay. Nucleotide diphosphate or monophosphate produced by the target enzyme displaces the tracer from the antibody, leading to a decrease in TR-FRET (Figure 1). The use of a red tracer minimizes interference from fluorescent compounds and light scattering. The Transcreener® TR-FRET Assays are designed specifically for HTS with a single addition, mix-and-read format.

A critical factor in realizing the advantages of the Transcreener® HTS assays is the correct setup of the microplate reader used for data readout. Proper selection of filters, dichroics, gain and flashes can impact the instrument`s sensitivity for any given assay. The key instrument parameters for Transcreener® HTS assay performance were identified by running a 10 μM ATP/ADP standard curve (24 replicates), as standard curves of this type mimic enzyme reactions. Starting with 10 μM ATP, ADP was added in increasing amounts and ATP is decreased proportionately, maintaining a total adenine nucleotide concentration of 10 μM. The flash numbers were varied to determine the requirements for a Z' > 0.5. In order to validate an instrument for use with the Transcreener® TR-FRET Assays, a Z' > 0.7 at 10% conversion of 10 μM ATP was required.

For more information, take a look at the following application notes:

Optimized Settings to Validate the BMG LABTECH PHERAstar Plus/FS HTS Microplate Readers with the Transcreener® TR-FRET Assays.

Optimized Settings to Validate the BMG LABTECH POLARstar/FLUOstar Omega Microplate Readers with the Transcreener® TR-FRET Assays.

The following BMG LABTECH microplate readers are Transcreener® ADP² TR-FRET certified from BellBrook Labs:

For more information about the Transcreener® ADP² TR-FRET Assay and other Transcreener® technologies visit:
http://www.bellbrooklabs.com