Thermo Scientific™

LanthaScreen™ Akt HEK 293E Cell Line

LanthaScreen™ GFP Cellular Assays allow for the analysis of post-translational modifications for a number of target proteins in an live-cell자세히 알아보기

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.

사양

대체 작용제Insulin

길항제LY294002, PI-103

어세이 입력Cell-based terbium phospho-specific activity (stable)

셀 상태Dividing Cells

검출 방법Fluorescence

유전자 기호AKT1

최적 반응IGF-1 (EC50 = 0.42 μg/mL)

일차 작용제IGF-1

제품라인LanthaScreen™

수량1 Vial

판독TR-FRET

리포터 유전자GFP (EmGFP)

타겟 입력AKT1

유형Cell Line

세포주293E (HEK)

System TypeLanthaScreen™

Druggable TargetSignaling Pathway

Unit SizeEach