Thermo Scientific™ LanthaScreen™ Akt HEK 293E Cell Line

Description

• LanthaScreen™ GFP Cellular Assays allow for the analysis of post-translational modifications for a number of target proteins in an live-cell context
• The PI3K/AKT pathway mediates signals for cell growth, cell survival, transcription, translation, and glucose uptake
• Because of the complexity of this signaling cascade, especially as applied to the regulation of the mammalian target of rapamycin (mTOR), cell-based methods are critical for proper identification of small-molecule mediators of this pathway
• The significance of mTOR as a kinase has been underscored recently by the identification of two distinct multimeric complexes inside the cell: mTORC1 (includes raptor and is rapamycin sensitive) and mTORC2 (includes rictor and is insensitive to acute rapamycin exposure)
• mTORC2 has been shown to phosphorylate and AKT at residue Ser473 for complete activation of this prosurvival kinase
• LanthaScreenTM AKT HEK293E is a human cell line which constitutively expresses GFP-AKT fusion proteins
• This kinase target was introduced using lipid transfection and these cells are a clonal population isolated by FACS, using GFP fluorescence as a sorting marker and Blasticidin to maintain cells under selection
• Using this cell line, a homogenous immunoassay was developed with a time-resolved FRET (TR-FRET) readout in which the insulin-induced phosphorylation of Ser473 on GFP-AKT is detected in cell lysates using a terbium-labeled phosphor-specific antibody
• This cell line has been validated with different stimuli/inhibitors and shows correct EC50/IC50 values
• Moreover, this assay has been optimized for performance under variable experimental conditions (including cell plating density, agonist stimulation time, DMSO tolerance and assay development time) and displays excellent statistical data (Z' > 0.6) and good signal-to-background.