This HTRF kit is designed to monitor the expression level of cellular AKT3 and can be used as a normalization assay for the phospho AKT3 kit.

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

This HTRF kit is designed to monitor the expression level of cellular AKT3 and can be used as a normalization assay for the phospho AKT3 kit.

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Overview

The Total AKT3 kit is designed to monitor the expression level of AKT3 and is used as a normalization assay with our phospho-AKT3 (Ser473) kits. 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. AKT3 in particular plays a critical role in neuroscience.

Total-AKT3 assay principle

The Total-AKT3 assay quantifies the expression level of AKT3 in a cell lysate. Contrary to Western Blot, the assay is entirely plate-based and does not require gels, electrophoresis or transfer. The Total-AKT3 assay uses two labeled antibodies: one coupled to a donor fluorophore, the other to an acceptor. Both antibodies are highly specific for a distinct epitope on the protein. In presence of AKT3 in a cell extract, the addition of these conjugates brings the donor fluorophore into close proximity with the acceptor and thereby generates a FRET signal. Its intensity is directly proportional to the concentration of the protein present in the sample, and provides a means of assessing the protein’s expression under a no-wash assay format.

Total-AKT3 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 Total AKT3 HTRF detection reagents. This protocol enables the cells' viability and confluence to be monitored.

Total-AKT3 1-plate assay protocol

Detection of total AKT3 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.

Total AKT3 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 sv white microplates and 2 µL of different concentration of blocking peptide specific for AKT3 was added before adding 4 µL of the HTRF total AKT3 detection reagents. The HTRF signal was recorded after an overnight incubation at room temperature.

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.
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

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 syqtem - 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

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 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

Product Insert AKT3 total Kit / 63ADK083PEG-63ADK083PEH

63ADK083PEG-63ADK083PEH - Product Insert

Cisbio Product Catalog 2019

All your HTRF assays in one document! - Catalog

A guide to Homogeneous Time Resolved Fluorescence

General principles of HTRF - Guides

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