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Thermo Scientific™

LanthaScreen™ PRAS40 293E Cell Line

Catalog number:  K1528

Related applications:

Pharma & Biopharma

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The PI3K/AKT pathway is activated by various stimuli (including insulin and IGF-1) and mediates signals for cell growth, cell survival, translation, and inhibition of apoptosis. PRAS40 (Proline-rich AKT substrate, 40 kDa) is a cytosolic protein that is phosphorylated at position Thr246 by AKT directly upon stimulation of the pathway. PRAS40 has been shown to bind to the mTORC1 complex and inhibit downstream signaling (i.e., phosphorylation of 4E-BP1 or p70S6K). LanthaScreen® PRAS40 HEK293E is a human cell line which constitutively expresses GFP-PRAS40 fusion proteins. The kinase substrate
was introduced using lentiviral transduction followed by selection with blasticidin. This cell line is a clonal population isolated by flow cytometry using GFP fluorescence as a sorting marker. The PI3K/AKT pathway is known to be active in the highly insulin-responsive HEK293E cell background. Using this cell line, a homogenous ('addition only') immunoassay has been developed in a 384-well format. The phosphorylation of PRAS40 at Thr246 is detected in cell lysates using a terbium-labeled phosphospecific antibody in a time-resolved FRET (TR-FRET) readout. The performance of this assay has been tested using numerous experimental variables, including cell plating density, stimulation time, DMSO tolerance, and antibody incubation time. Under optimized conditions, the assay has been further validated and shows correct EC50 and IC50 values, with insulin as the primary agonist and PI-103 as the known inhibitor. Overall, the assay displays excellent statistical data (Z'-factor >0.6), high signal-to-background (response ratio), and is a robust cell-based readout of AKT signaling.
For Research Use Only. Not for use in diagnostic procedures.


Alternate Agonists: Insulin
Antagonists: IGF-1
Assay Entry: Cell-based terbium phospho-specific activity (stable)
Cell State: Dividing Cells
Druggable Target: Kinases, Signaling Pathway
Gene Symbol: PRAS40
Primary Agonist: Insulin
Product Line: LanthaScreen®
Readout: End Point
Reporter Gene: GFP (EmGFP)
System Type: LanthaScreen®
Target Entry: PRAS40

Contents & storage

LanthaScreen® PRAS40 HEK293E cells are shipped on dry ice. Store in liquid nitrogen immediately upon receipt, or thaw for immediate use.