Ras Activation Redistribution Assay

• Designed to assay compounds for their ability to modulate activation of Ras
• Coupled to EGFP for easy monitoring of the cellular translocation event
• Robust cell-based assay for use in high content analysis and fluorescence microscope applications

• Biologically relevant data: Compounds tested in a cellular environment
• Validated: Functionally tested cells provided with an optimized assay protocol
• Easy to use: Just plate cells, add compounds, and image

GTP-bound Ras activates the Raf/MEK/ERK pathway by recruiting Raf to the plasma membrane. This leads to 14-3-3 protein displacement from the N-terminus of Raf and de-phosphorylation of an otherwise protected negative regulatory site. Ras/Raf interaction is dependent on Ras:GTP binding. This has lead to the use of the Ras-binding domain of Raf (RafRBD) fused to glutathione S-transferase (GST) as a capture agent for analyzing the activation state of Ras. The Ras activation Redistribution assay employs RafRBD fused to GFP as a read-out for Ras activity. Active Ras recruits RafRBD-EGFP to the plasma membrane whereas inactive Ras leaves RafRBD-EGFP free in the cytoplasm.

Figure 1. Translocation of RafRBD-EGFP. Cells were untreated (DMSO control, left panel), treated with 100 nM insulin (center panel), or treated with 100 nM IGF-1  (right panel) for 5 min. Arrows indicate the membrane localization detected by the image analysis algorithm.

Figure 2. IGF-1 concentration response in the Ras Activation Redistribution® assay. Concentration response was measured in 9 point half log dilution series (n=16). Cells were incubate d with IGF-1 for 5 min. Cells were then fixed and the cytoplasm to membrane translocation was measured using the Cellomics ArrayScan V^TI Reader and the CytoCellMemTrans.V2 BioApplication. % activity was calculated relative to the positive (100 nM IGF-1) and negative control (0.25% DMSO). The EC₅₀ of IGF-1 is ~0.5 nM.