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HTRF Human Phospho-AKT1/2/3 (Thr308) Detection Kit, 500 Assay Points

This HTRF kit is designed to monitor pan AKT phosphorylation on Thr308 as a readout of the activated Pi3K pathway.

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: 64AKTPEG
Unit Size: 500 Assay Points
Part Number: 64AKTPEH
Unit Size: 10,000 Assay Points
  • The Phospho-AKT (Thr308) kit is designed for robust quantification of AKT modulation, phosphorylated on Thr308 across all isoforms (AKT1/2/3). Also known as protein kinase B (PKB), AKT is an oncogene playing a key role in controlling apoptosis, cell proliferation, transcription, cell migration, and glucose metabolism.

  • Assay Points
    500
    Assay Target Type
    Kit
    Assay Technology
    HTRF
    Brand
    HTRF
    Quantity
    1
    Therapeutic Area
    Metabolism/Diabetes
    NASH/Fibrosis
    Oncology & Inflammation
    Unit Size
    500 Assay Points
  • Phospho-AKT (Thr308) assay principle

    The Phospho-AKT (Thr308) assay measures AKT when phosphorylated at Thr308. Contrary to Western Blot, the assay is entirely plate-based and does not require gels, electrophoresis or transfer. The Phospho-AKT (Thr308) 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.

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    Phospho-AKT (Thr308) 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 (Thr308) HTRF detection reagents. This protocol enables the cells' viability and confluence to be monitored.

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    Phospho-AKT (Thr308) 1-plate assay protocol

    Detection of Phosphorylated AKT (Thr308) 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.

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  • Phospho-AKT (Thr308) IGF dose-response on CHO-M1 cells

    Results obtained on CHO cells plated using 2 cell densities (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 on Thr308 was measured after 3.5 hours using the two-plate assay protocol.

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  • 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.

    2phospho-pathway-akt-63adk078peg-63adk078peh-63adk078pey.svg

     

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