Phospho-AKT1/2/3 (Ser473) cellular kit
Simple, all-in-one kit for robust detection of Phospho-ATK & Total ATK
Total AKT cellular kit HTRF®
This HTRF kit detects cellular AKT and can be used as a normalization assay for the phospho-AKT kits.
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Highly specific
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Ease-of-use
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High sensitivity
Overview
The Total ATK kit is a robust, ready-to-use assay designed to monitor the modulation in expression of all 3 isoforms of AKT, an oncogene controlling apoptosis, cell proliferation, transcription, cell migration, and glucose metabolism. This kit is the ideal companion for our phospho AKT (Ser473 and Thr 308) kits, and is used as a normalization assay.
This kit has been pre-validated for use in combination with Pelago Bioscience’s CETSA® (Cellular Thermal Shift Assay) technology. Please note that further assay optimization will be required in order to adapt the kit to your particular cell line and experimental conditions. A valid CETSA® licence from Pelago Bioscience or a single kit agreement is required for using the CETSA® technology.
Benefits
- VALIDATED ON MULTIPLE CELL TYPES
- SPECIFICITY
- DATA NORMALIZATION
Total-AKT assay principle
The Total-AKT assay quantifies the expression level of AKT in a cell lysate. Contrary to Western Blot, the assay is entirely plate-based and does not require gels, electrophoresis or transfer. The Total-AKT 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 AKT 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 proteins expression under a no-wash assay format.
Total-AKT 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 AKT HTRF detection reagents. This protocol enables the cells' viability and confluence to be monitored.
Total-AKT 1-plate assay protocol
Detection of total AKT 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 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.
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
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
HTRF products and supporting scientific content to investifgate GPCRs - Flyers
Cell Signaling: Biomarkers, Phospho- & total-protein assays - Flyers
HTRF assays for Oncology and Inflammation
Signaling in the Immune System - Brochures
In collaboration with Molecular Devices - Posters
PI3K/AKT/mTor translational control pathway - Posters
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
From 2D, 3D cell cultures to xenografts: A smart HTRF platform to maximize anticancer drug discovery
One technology across all samples - Application Notes
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
Multi-tissue cellular modeling and anlysis of insulin signaling - Posters
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
A single technology for 2D cells, 3D cells, and xenograft models - 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
Cell-based kinase assays in HTS ? potential and limitations for primary and secondary screening
In collaboration with Bayer - Scientific Presentations
Manual AKT total Kit / 64NKTPET
64NKTPET - Product Insert
STING HTRF offer to bridge innate and adaptive immunity
cGAS-STING signaling pathway from A to Z - Brochures
Manual AKT total Kit / 64NKTPEG-64NKTPEH
64NKTPEG-64NKTPEH - Product Insert
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
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
Safety Data Sheet (DEU) AKT total Kit / 64NKTPEG
64NKTPEG - Safety Data Sheet
Safety Data Sheet (ELL) AKT total Kit / 64NKTPEG
64NKTPEG - Safety Data Sheet
Safety Data Sheet (FRA-FR) AKT total Kit / 64NKTPEG
64NKTPEG - Safety Data Sheet
Safety Data Sheet (ITA) AKT total Kit / 64NKTPEG
64NKTPEG - Safety Data Sheet
Safety Data Sheet (SPA) AKT total Kit / 64NKTPEG
64NKTPEG - Safety Data Sheet
Safety Data Sheet (ENG-GB) AKT total Kit / 64NKTPEG
64NKTPEG - Safety Data Sheet
Safety Data Sheet (ENG-US) AKT total Kit / 64NKTPEG
64NKTPEG - Safety Data Sheet
Certificate of Analysis AKT total Kit / 64NKTPEH
64NKTPEH Batch 11E - Quality Control Report
Safety Data Sheet (DEU) AKT total Kit / 64NKTPEH
64NKTPEH - Safety Data Sheet
Safety Data Sheet (ELL) AKT total Kit / 64NKTPEH
64NKTPEH - Safety Data Sheet
Safety Data Sheet (FRA-FR) AKT total Kit / 64NKTPEH
64NKTPEH - Safety Data Sheet
Safety Data Sheet (ITA) AKT total Kit / 64NKTPEH
64NKTPEH - Safety Data Sheet
Safety Data Sheet (SPA) AKT total Kit / 64NKTPEH
64NKTPEH - Safety Data Sheet
Safety Data Sheet (ENG-GB) AKT total Kit / 64NKTPEH
64NKTPEH - Safety Data Sheet
Safety Data Sheet (ENG-US) AKT total Kit / 64NKTPEH
64NKTPEH - Safety Data Sheet
Certificate of Analysis AKT total Kit / 64NKTPET
64NKTPET Batch 01T - Quality Control Report
Safety Data Sheet (DEU) AKT total Kit / 64NKTPET
64NKTPET - Safety Data Sheet
Safety Data Sheet (ELL) AKT total Kit / 64NKTPET
64NKTPET - Safety Data Sheet
Safety Data Sheet (FRA-FR) AKT total Kit / 64NKTPET
64NKTPET - Safety Data Sheet
Safety Data Sheet (ITA) AKT total Kit / 64NKTPET
64NKTPET - Safety Data Sheet
Safety Data Sheet (SPA) AKT total Kit / 64NKTPET
64NKTPET - Safety Data Sheet
Safety Data Sheet (ENG-GB) AKT total Kit / 64NKTPET
64NKTPET - Safety Data Sheet
Safety Data Sheet (ENG-US) AKT total Kit / 64NKTPET
64NKTPET - Safety Data Sheet
Certificate of Analysis AKT total Kit / 64NKTPET
64NKTPET Batch 01V - Quality Control Report
Safety Data Sheet (DEU) AKT total Kit / 64NKTPEY
64NKTPEY - Safety Data Sheet
Safety Data Sheet (ELL) AKT total Kit / 64NKTPEY
64NKTPEY - Safety Data Sheet
Safety Data Sheet (FRA-FR) AKT total Kit / 64NKTPEY
64NKTPEY - Safety Data Sheet
Safety Data Sheet (ITA) AKT total Kit / 64NKTPEY
64NKTPEY - Safety Data Sheet
Safety Data Sheet (SPA) AKT total Kit / 64NKTPEY
64NKTPEY - Safety Data Sheet
Safety Data Sheet (ENG-GB) AKT total Kit / 64NKTPEY
64NKTPEY - Safety Data Sheet
Safety Data Sheet (ENG-US) AKT total Kit / 64NKTPEY
64NKTPEY - Safety Data Sheet
Plate Reader Requirement
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