TR-FRET (Time-Resolved Fluorescence Energy Transfer) is a special form of FRET (Förster Resonance Energy Transfer) in which the long-lived emission fluorophores are used as donors.
For the investigation of protein interactions, methods have been established which exploit the energy transfer from fluorescence light to a second fluorophore (FRET). This only takes place when two molecules are in close proximity, for example when two proteins interact. This requires two proteins that are coupled to different fluorophores: a donor that emits light after excitation and only excites the acceptor fluorophore when the bound proteins interact. The ratio of acceptor to donor fluorescence provides information on the association of both proteins.
Time Resolved FRET Cellular Kinase Assay
The TR-FRET Cellular Kinase Assay Kits are intended for the simple and rapid detection of either phosphorylated or total protein in cell lysates using the homogeneous (no-wash) TR-FRET technology. Cells are first treated to modulate protein levels. Cells are then lysed with the specific lysis buffer provided in the kit and the target protein is subsequently detected in one-step using the TR-FRET reagents.
The target protein (phosphorylated or total) is detected in a sandwich assay format using 2 different labelled specific antibodies.
The transmitter of the fluorescence emission is preferably made up of molecules with a long emission lifetime (time resolved FRET, TR-FRET). This allows the signal to be detected at a later point in time and over a longer period of time and minimizes background fluorescence.
Excitation of the donor with a flash lamp (320 or 340 nm) or a laser (337 nm) triggers a Fluorescence Resonance Energy Transfer (FRET) towards the acceptor, which in turn emits light at 665 nm. The signal at 665 nm is proportional to the concentration of protein in the cell lysate.
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