Transcreener ADP2 TR-FRET assay

Transcreener® assays rely on direct, highly-specific detection of nucleotides using antibodies that are able to differentiate between nucleotides on the basis of a single phosphate group. These assays can be used across entire families of nucleotide-dependent enzymes. The Transcreener assays use a homogenous, competitive immunoassay format in which the antibodies are paired with high affinity fluorescent tracers. Displacement of the tracer by the nucleotide being detected causes a change in its fluorescence properties.

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 complex in the UV range (approx. 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. 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 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. 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 is required.

Application note

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

 

Transcreener is a patented technology of BellBrook Labs.

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