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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 and disposal requirements under European REACH regulations (EC 1907/2006).

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Feature Specification
Application Cell Signaling
Sample Volume 16 µL

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 and disposal requirements under European REACH regulations (EC 1907/2006).

Click to copy promo code to clipboard.
SAVE 15% NOW on online orders. Use promo code below.
YEAREND15
Offer valid until 12/15. Terms and conditions apply.
Product Variants
Unit Size: 500 Assay Points
Part #:
64AKTPEG
List Price
USD 2,147.00
Unit Size: 10,000 Assay Points
Part #:
64AKTPEH
List Price
USD 12,490.00

Overview

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.

Specifications

Application
Cell Signaling
Brand
HTRF
Detection Modality
HTRF
Lysis Buffer Compatibility
Lysis Buffer 1
Lysis Buffer 4
Lysis Buffer 5
Molecular Modification
Phosphorylation
Product Group
Kit
Sample Volume
16 µL
Shipping Conditions
Shipped in Dry Ice
Target Class
Phosphoproteins
Target Species
Human
Technology
TR-FRET
Therapeutic Area
Metabolism/Diabetes
NASH/Fibrosis
Oncology & Inflammation
Unit Size
500 Assay Points

Video gallery

Citations

How it works

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.

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

 

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.

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

 

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.

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

 

Assay validation

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.

assay-validation-akt-phospho-t308.svg

 

Simplified pathway

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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