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LanthaScreen® Akt HEK 293E Cell Line

Catalog number: K1615

 Related applications: Target & Lead Identification & Validation

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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.
For Research Use Only. Not for use in diagnostic procedures.

Specifications

System: LanthaScreen®
Readout: TR-FRET
Reporter: GFP (EmGFP)
Cell Line: 293E (HEK)
Cell State: Dividing Cells
Antagonists: PI-103, LY294002
Gene Symbol: AKT1
Product Size: 1 vial
Primary Agonist: IGF-1
Detection Method: Fluorescent
Druggable Target: Kinases, Signaling Pathway
Optimal Response: IGF-1 (EC50 = 0.42 µg/ml)
Alternate Agonists: Insulin

Contents & storage

LanthaScreen® AKT HEK 293E cells are shipped on dry ice. Store in liquid nitrogen immediately upon receipt, or thaw for immediate use.

Documents

Manuals & protocols