Time Resolved-Fluorescence Resonance Energy Transfer (TR-FRET) is a preferred fluorescent assay format in drug discovery laboratories. TR-FRET assays are less susceptible to compound interference than other assay formats and may be applied to multiple target classes. To support this technology focus, Life Technologies has developed LanthaScreenTM biochemical and cellular assays to measure kinase activity, compound binding, and post-translation modification events, such as phosphorylation and acetylation of substrates, in cell signaling. LanthaScreenTM assays utilize terbium and europium chelates as FRET donors. Terbium chelate is used as a donor for fluorescein and GFP and europium chelate is a donor for Alexa Fluor® 647.
The LanthaScreen™ kinase activity assay format is based on the use of a long-lifetime terbium or europium chelate as the donor species and fluorescein as the acceptor species. When terbium- and fluorescein-labeled molecules are brought into proximity, energy transfer takes place causing an increase in acceptor fluorescence and a decrease in donor fluorescence. The TR-FRET value is determined as a ratio of the FRET-specific signal measured with a 520 nm filter to that of the signal measured with a 495 nm filter, which is specific to terbium.
Fig. 1: Schematic of LanthaScreen® kinase activity assay using traditional peptide substrates. A fluorescein-labeled kinase substrate peptide is incubated with kinase and ATP. Terbium or europium-labeled antibody is then added and phosphorylation detected by an increase in the TR-FRET value. Because the substrate is directly labeled, there is no need to add streptavidin-APC. Additionally, unlike other europium-based systems, there is no requirement to add high concentrations of potassium fluoride, which can potentially disrupt antibody–product interactions.
In a LanthaScreen kinase assay, kinase, fluorescein-labeled substrate, and ATP are allowed to react. Then EDTA (to stop the reaction) and terbium-labeled antibody (to detect phosphorylated product) are added. In a LanthaScreen kinase reaction, the antibody associates with the phosphorylated fluorescein labeled substrate resulting in an increased TR-FRET value. The amount of antibody that is bound to the tracer is directly proportional to the amount of phosphorylated substrate present, and in this manner, kinase activity can be detected and measured by an increase in the TR-FRET value.
Fig. 2: Schematic of LanthaScreen® cellular assay
The principle and methods for this assay format are simple:
- 1. Phosphorylation of the kinase substrate expressed as a GFP-fusion protein is either stimulated with an agonist to activate the pathway or treated with an inhibitor to interrupt the signaling event. No protein modification occurs in unstimulated cells.
- 2. Cells are lysed in a buffer that contains a Tb-labeled phosphorylation site-specific antibody. The detection of the protein modification event is measured on a TR-FRET-compatible plate reader. Little or no TR-FRET is observed with unstimulated or inhibited cells whereas stimulated cell samples display high TR-FRET.
See our LanthaScreen application notes:
For more information about LanthaScreen® and related assays available from Life Technologies, please visit their website.