This HTRF kit is designed to monitor AKT1 phosphorylation on Ser473 as a readout of Pi3K pathway activation.

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  • Highly specific Highly specific
  • Ease-of-use Ease-of-use
  • High sensitivity High sensitivity

This HTRF kit is designed to monitor AKT1 phosphorylation on Ser473 as a readout of Pi3K pathway activation.

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Overview

The phospho-AKT1 (Ser473) kit is designed for robust quantification of AKT1 modulation, phosphorylated on Ser473. 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. AKT1 in particular plays a critical role in cell survival, and thus has important applications in cancer research.

Benefits

  • VALIDATED WITH BLOCKING PEPTIDE
  • SPECIFICITY

Phospho-AKT1 (Ser473) assay principle

The Phospho-AKT1 (Ser473) assay measures AKT1 when phosphorylated at Ser473. Contrary to Western Blot, the assay is entirely plate-based and does not require gels, electrophoresis or transfer. The Phospho-AKT1 (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-AKT1 (Ser473) assay principle

Phospho-AKT1 (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-AKT1 (Ser473) HTRF detection reagents. This protocol enables the cells' viability and confluence to be monitored.
Phospho-AKT1 (Ser473) 2-plate assay protocol

Phospho-AKT1 (Ser473) 1-plate assay protocol

Detection of Phosphorylated AKT1 (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-AKT1 (Ser473) 1-plate assay protocol

Phospho AKT1 assay: Inhibitory effect of blocking peptide

SH-SY5Y cells were stimulated with human IGF-1 for 15 min. After stimulation, media was removed and cells were lysed with lysis buffer 1X for 30 min at RT under gentle shaking. 14 µL of lysates were transferred into 384-well small volume white microplates and 2 µL of different concentration of blocking peptide specific for AKT1 was added before adding 4 µL of the HTRF phospho AKT1 (Ser473) detection reagents. The HTRF signal was recorded after an overnight incubation at room temperature.
Phospho AKT1 assay: Inhibitory effect of blocking peptide

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 - Notes d'application

HTRF: One technology, many uses in a pre-clinical laboratory setting

Benefits and considerations of HTRF - Présentations scientifiaues

Cisbio lysis buffer compatibility

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

HTRF cellular phospho-protein assays

physiologically relevant results fo fast flowing research - Flyers

GPCR research from A to Z

HTRF products and supporting scientific content to investifgate GPCRs - Flyers

Save time and money

Switch to HTRF assays - Flyers

Inflammation cell by cell

HTRF solutions for each cell type - Flyers

Species compatibility

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

HTRF assays for Oncology and Inflammation

Signaling in the immune system - Brochures

Side-by-side comparison of HTRF, Western Blot, ELISA and AlphaScreen® SureFire®

Do all cell-based kinase assays perform similarly? - Posters

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

New cell-based HTRF® assays for the exploration of Wnt signaling pathway

Investigation of the endogenous AKT/GSK3 and Wnt pathways - Posters

TCR signaling investigation with HTRF phospho assays

Study a pathway of interest in PBMC and T cells - Notes d'application

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

Tumor xenograft analysis: HTRF versus Western blot - Notes d'application

HTRF cell-based phospho-protein data normalization

Valuable guidelines for efficiently analyzing and interpreting results - Notes d'application

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

Increased flexibility of phospho-assays - Notes d'application

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

Insider Tips for successful sample treatment - Notes techniques

HTRF Alpha-tubulin Housekeeping kit

Properly interpret your compound effect - Notes d'application

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 - Notes techniques

Key guidelines to successful cell signaling experiments

Mastering the art of cell signaling assays optimization - Guides

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 - Notes techniques

How to run a cell based phospho HTRF assay

What to expect at the bench - Vidéos

Unleash the potential of your phosphorylation research with HTRF

Unmatched ease of use, sensitivity and specificity assays - Vidéos

STING HTRF offer to bridge innate and adaptive immunity

cGAS-STING signaling pathway from A to Z - Brochures

Cisbio Product Catalog 2019

All your HTRF assays in one document! - Catalogue

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

HTRF® cell signaling platform combined with iCell® Hepatocytes

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

Unleash the potential of your phosphorylation research with HTRF

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

How to run a cell based phospho HTRF assay

3' video to set up your Phospho assay - Vidéos

Molecular basis of neuroinflammation and neurodegeneration diseases

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

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