LanthaScreen Cellular Assays

• Screen quickly with a simple and homogenous high-throughput screening (HTS) – amenable assay format
• Interrogate the effects of your compounds on specific signaling events within a pathway
• Analyze multiple post-translational modifications using physiological pathway kinetics

Assay Principle

The cellular response to environmental cues is mediated through a complex network of signal transduction pathways.  Alteration or disruption of components within these pathways often contributes to various diseases, including cancer and metabolic and inflammatory disorders.  The diversity of validated drug targets in these signal transduction cascades provides both challenges and opportunities in drug discovery.  Intermediate signaling events, such as phosphorylation and ubiquitination, are bona fide targets for assay development.  Until now, there have been a limited number of HTS-compatible methods available to interrogate, in a cell-based format, the regulated activity of enzymes responsible for posttranslational modifications.

Combining the simplicity of biochemical assays with the intricacy of cellular systems, Invitrogen has developed time-resolved fluorescence resonance energy transfer (TR-FRET)-based assays to monitor protein modification within the native cell signaling environment.  By expressing the substrate of interest as a fusion with green fluorescent protein (GFP), modification-specific antibodies labeled with the TR-FRET donor partner terbium (Tb) can be used to quantitatively monitor these modifications following cell lysis (Figure 1).

Figure 1—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.