Akt1-PH Redistribution Assay

• Designed to assay compounds for their ability to modulate membrane translocation of the Akt1-PH domain
• CouplAed 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

The Akt1/PKBα signaling pathway is activated by various growth factors (e.g. insulin, IGF-I) and mediates cell survival signals. Upon activation of the pathway, Akt1 translocates from the cytoplasm to the plasma membrane, due to the production of specific phospholipids by phosphatidylinositol 3-kinase (PI3K) in the membrane. The Akt1-PH domain Redistribution assay monitors translocation of the EGFP-Akt1-PH fusion protein from the cytoplasm to the plasma membrane. The assay is designed as a companion assay to be used in combination with our full length Akt1 Redistribution assay (see related products) to elucidate the significance of the PH-domain of Akt1 for small molecule inhibitors of Akt1 membrane translocation.

Figure 1. Membrane translocation of EGFP-Akt1-PH. Cells were treated with 100 nM IGF-1 without (DMSO control, left panel) and with addition of 500 nM wortmannin (right panel). Arrows indicate IGF-1 induced membrane translocation of EGFP-Akt1-PH detected by the image analysis algorithm.

Figure 2. Concentration response curve in the Akt1-PH antagonist assay. Wortmannin concentration response curve in the Akt1-PH antagonist Redistribution assay stimulated by 100 nM IGF-1 (n=16). The EC₅₀ of wortmannin is 105 nM. Concentration response was measured in 9 point half log dilution series. Cells were pre-incubated with 100 nM IGF-1 for 60 min. and treated with wortmannin for 4 min. Cells were then fixed and membrane translocation was measured using the Cellomics ArrayScan® V^TI Reader and the CytoCellMemTrans.V2 BioApplication. % activity was calculated relative to the positive (500 nM wortmannin) and negative control (0.25% DMSO).