Skip to main content

HTRF Human Phospho-AKT1/2/3 (Ser473) Detection Kit, 96 Assay Points

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

For research use only. Not for use in diagnostic procedures. All products to be used in accordance with applicable laws and regulations including without limitation, consumption & disposal requirements under European REACH regulations (EC 1907/2006).
Part Number: 64AKSPET
Unit Size: 96 Assay Points
Part Number: 64AKSPEG
Unit Size: 500 Assay Points
Part Number: 64AKSPEH
Unit Size: 10,000 Assay Points
  • 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.

  • Assay Points
    96
    Assay Target Type
    Kit
    Assay Technology
    HTRF
    Brand
    HTRF
    Quantity
    1
    Unit Size
    96 Assay Points
  • 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 proteins phosphorylation state under a no-wash assay format.

    phospho-how-it-works-phospho-btk-tyr223-a1.svg

     

    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.

    2biomarkers-how-it-works-assay-protocol-alpha-tubulin-63adk072peg-63adk072peh.svg

     

    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.

    2phospho-how-it-works-total-btk-1-plate-assay-protocol.svg

     

  • 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.

    assay-validation-akt-phospho-s473-1.svg

     

    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.

    assay-validation-akt-phospho-s473-2.svg

     

  • 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.

    phospho-pathway-akt-64akspeg-64akspeh-64akspet-64akspey3.svg

     

Resources

1-2 of 2 Resources
Guide
HTRF solutions, guide to major applications

This guide provides you an overview of HTRF applications in several therapeutic areas.

Flyer
Reagent solutions for autoimmunity research.

Advance your autoimmune disease research and benefit from Revvity broad offering of reagent technologies