Phospho-AKT1/2/3 (Ser473) cellular kit HTRF®

This HTRF kit is designed to monitor pan Akt phosphorylation on Ser473 as a readout of Pi3K pathway activation.

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  • High sensitivity High sensitivity

This HTRF kit is designed to monitor pan Akt phosphorylation on Ser473 as a readout of Pi3K pathway activation.



The Phospho-AKT (Ser473) kit is designed to robustly and efficiently monitor AKT phosphorylation on Ser473 across all isoforms, as a readout of Pi3K pathway activation. Also known as Protein Kinase B (PKB), AKT is an oncogene that controls apoptosis, cell proliferation, transcription, cell migration, and glucose metabolism.



Phospho-AKT (Ser473) assay principle

The Phospho-AKT (Ser473) assay measures AKT when phosphorylated at Ser473. Contrary to Western Blot, the assay is entirely plate-based and does not require gels, electrophoresis or transfer. The Phospho-AKT (Ser473) assay uses 2 labeled antibodies: one with a donor fluorophore, the other one with an acceptor. The first antibody is selected for its specific binding to the phosphorylated motif on the protein, the second for its ability to recognize the protein independent of its phosphorylation state. Protein phosphorylation enables an immune-complex formation involving both labeled antibodies and which brings the donor fluorophore into close proximity to the acceptor, thereby generating a FRET signal. Its intensity is directly proportional to the concentration of phosphorylated protein present in the sample, and provides a means of assessing the protein’s phosphorylation state under a no-wash assay format.
Phospho-AKT (Ser473) assay kit principle

Phospho-AKT (Ser473) 2-plate assay protocol

The 2 plate protocol involves culturing cells in a 96-well plate before lysis then transferring lysates to a 384-well low volume detection plate before adding Phospho-AKT (Ser473) assay HTRF detection reagents. This protocol enables the cells' viability and confluence to be monitored.
Phospho-AKT (Ser473) assay kit 2-plate assay protocol

Phospho-AKT (Ser473) assay 1-plate assay protocol

Detection of Phosphorylated AKT (Ser473)with HTRF reagents can be performed in a single plate used for culturing, stimulation and lysis. No washing steps are required. This HTS designed protocol enables miniaturization while maintaining robust HTRF quality.
Phospho-AKT (Ser473) assay kit 1-plate protocol

Phospho-AKT (Ser473) Assay IGF Dose-Response on HEK 293 cells

Results obtained on HEK cells plated using 3 cell densities (12,500 - 25,000 - 50,000 cells per well). After being starved for 24 hours, cells were stimulated for 10 minutes with various concentrations of IGF. Phosphorylated AKT was measured after 3.5 hours using the two-plate assay protocol.
Phospho-AKT (Ser473) Assay IGF Dose-Response on HEK 293 cells

Total AKT used to control the phosphorylation status of AKT (Ser473).

HEK293 cells (100,000 cells/well) were activated with IGF-1 for 10 min, using the two-plate assay protocol of the Phospho-AKT (Ser473) and Total-AKT assays. As expected, results obtained show a dose-response increase of AKT phosphorylation upon IGF-1 stimulation, while the AKT expression level remains constant.
Dose response of Phospho (S473) and Total AKT upon IGF-1 stimulation

AKT Simplified Pathway

AKT (or protein kinase B) plays a key role in controlling survival and apoptosis. This serine/threonine protein kinase is regulated by insulin and various growth and survival factors, to work in a wortmannin-sensitive pathway involving PI 3 kinase. When the Pleckstrin Homology (PH) domain of AKT binds to phosphoinositides, AKT can be phosphorylated by two different kinases, PDK1 at threonine 308 and mTORC2 (mammalian target of rapamycin) at serine 473, which switch on AKT activation.
AKT signaling pathway

Simplified pathway dissection with HTRF phospho-assays and CyBi-felix liquid handling

Analyse of PI3K/AKT/mTor translational control pathway - Application Notes

Open R&D: Sanofi Access Platform

In collaboration with Sanofi - Scientific Presentations

Lysis buffer compatibility

Cell Signaling: Biomarkers, Phospho- & total-protein Assays - Flyers

HTRF cellular phospho-protein assays

Physiologically relevant results fo fast flowing research - Flyers

Biomarker and Cell Signaling Assays for Fibrosis and NASH

HTRF and Alpha solutions for NASH - Flyers

GPCR research from A to Z

HTRF products and supporting scientific content to investifgate GPCRs - Flyers

Species compatibility

Cell Signaling: Biomarkers, Phospho- & total-protein assays - Flyers

HTRF assays for Oncology and Inflammation

Signaling in the Immune System - Brochures

Deciphering the mechanism of action of drugs targeting EGFR, by Cisbio's advanced cell-based pathway readout assays.

mechanism of action of EGFR inhibitors assessed in the pancreatic BXPC3 cell line - Posters

Universal HTRF® phospho-protein platform: from 2D, 3D, primary cells to patient derived tumor cells

Analysis of a large panel of diverse biological samples and cellular models - Posters

HTRF phospho assays reveal subtle drug induced effects in tumor-xenografts

Tumor xenograft analysis: HTRF versus Western blot - Application Notes

HTRF cell-based phospho-protein data normalization

Valuable guidelines for efficiently analyzing and interpreting results - Application Notes

HTRF phospho-total lysis buffer: a universal alternative to RIPA lysis buffers

Increased flexibility of phospho-assays - Application Notes

Best practices for analyzing brain samples with HTRF® phospho assays for neurosciences

Insider Tips for successful sample treatment - Technical Notes

HTRF Alpha-tubulin Housekeeping kit

Properly interpret your compound effect - Application Notes

Understanding GPCRs is the key to improved DD

In collaboration with GEN - Guides

Optimize your HTRF cell signaling assays on tissues

HTRF and WB compatible guidelines - Technical Notes

Key guidelines to successful cell signaling experiments

Mastering the art of cell signaling assays optimization - Guides

HTRF® cell signaling platform combined with iCell® Hepatocytes

A solution for phospho-protein analysis in metabolic disorders - Posters

HTRF phospho-assays reveal subtle drug-induced effects

Detailed protocol and direct comparison with WB - Posters

Best practices for analyzing tumor xenografts with HTRF phospho assays

Protocol for tumor xenograft analysis with HTRF - Technical Notes

How to run a cell based phospho HTRF assay

What to expect at the bench - Videos

Unleash the potential of your phosphorylation research with HTRF

Unmatched ease of use, sensitivity and specificity assays - Videos

STING HTRF offer to bridge innate and adaptive immunity

cGAS-STING signaling pathway from A to Z - Brochures

HTRF Product Catalog

All your HTRF assays in one document! - Catalog

A guide to Homogeneous Time Resolved Fluorescence

General principles of HTRF - Guides

How HTRF compares to Western Blot and ELISA

Get the brochure about technology comparison. - Brochures

Unleash the potential of your phosphorylation research with HTRF

A fun video introducing you to phosphorylation assays with HTRF - Videos

How to run a cell based phospho HTRF assay

3' video to set up your Phospho assay - Videos

Molecular basis of neuroinflammation and neurodegeneration diseases

The essential guide for extending your knowledge on the molecular mechanisms of neurodegenerative diseases - Guides

Neurodegeneration and its main related diseases

Discover this infographic design on neurodegenerative diseases - Infographics

On-demand webinar: Linking Neuroinflammation and Neurodegeneration

New insight into neuroinflammation research - Videos

Assays for autoimmunity research

Advance your research on autoimmune diseases - Flyers

Assays for neurosciences research

Advance your research on neurodegenerative diseases - Flyers

Inhibition of FGFR Signaling Pathways Studied in Cancer Cell Lines Using HTRF

The knowledge and data necessary to understand the inhibition value of FGFR receptors involved in human cancers. HTRF technology enables the monitoring of the activity of the AZD4547 inhibitor on cell signal transduction in 3 cancer cell lines. - Application Notes

Advance your research on Fibrosis

Kits and reagents for Fibrosis research - Flyers

Signaling pathways in liquid tumors

This litterature review highlights the signaling pathways and give examples of how HTRF® KinEASE™ and AlphaLISA® SureFire® assays can be used in the development of novel therapies for leukemia treatment - Application Notes

Guidelines for Cell Culture and Lysis in Different Formats Prior to HTRF Detection

Seeding and lysing recommendations for a number of cell culture vessels. - Technical Notes

Assessment of drug efficacy and toxicity by combining innovative technologies

Combination of AlphaLISA®, HTRF®, or AlphaLISA® SureFire® Ultra™ immunoassays with the ATPlite™ 1step cell viability assay - Application Notes

Methodological Aspects of Homogeneous Time-Resolved Fluorescence (HTRF)

Learn how to reduce time and sample consumption - Application Notes

Manual AKT P-S473 Kit / 64AKSPEG-64AKSPEH

64AKSPEG-64AKSPEH - Product Insert

Manual AKT P-S473 Kit / 64AKSPET

64AKSPET - Product Insert

Certificate of Analysis AKT P-S473 Kit / 64AKSPEG

64AKSPEG Batch 19K - Quality Control Report

Certificate of Analysis AKT P-S473 Kit / 64AKSPEG

64AKSPEG Batch 19L - Quality Control Report

Certificate of Analysis AKT P-S473 Kit / 64AKSPEG

64AKSPEG Batch 19M - Quality Control Report

Certificate of Analysis AKT P-S473 Kit / 64AKSPEG

64AKSPEG Batch 19N - Quality Control Report

Certificate of Analysis AKT P-S473 Kit / 64AKSPEG

64AKSPEG Batch 19P - Quality Control Report

Certificate of Analysis AKT P-S473 Kit / 64AKSPEH

64AKSPEH Batch 21C - Quality Control Report

Certificate of Analysis AKT P-S473 Kit / 64AKSPET

64AKSPET Batch 19G - Quality Control Report

Certificate of Analysis AKT P-S473 Kit / 64AKSPET

64AKSPET Batch 19H - Quality Control Report

Plate Reader Requirement

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