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HTRF Human Phospho-AKT (Ser473) Detection Kit, 500 Assay Points

The HTRF Human Phospho-AKT (Ser473) Detection Kit is an immunoassay designed for the detection and quantitation of Human AKT when phosphorylated at Ser473 in cellular lysates, in a homogeneous (no-wash steps, no separation steps) format.

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Feature	Specification
Application	Cell Signaling
Sample Volume	16 µL

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Product variants

Part #:

64AKSPET

List price

USD 724.00

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Part #:

64AKSPEG

List price

USD 2,340.00

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Part #:

64AKSPEH

List price

USD 13,611.00

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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).

Product information

Overview

The Phospho-AKT (Ser473) kit is designed to robustly and efficiently monitor AKT phosphorylation on Ser473 across all isoforms, as a readout of Pi3K pathway activation. Also known as Protein Kinase B (PKB), AKT is an oncogene that controls apoptosis, cell proliferation, transcription, cell migration, and glucose metabolism.

HTRF assays offer many advantages over other technologies:

Homogeneous add-and-read format
No wash steps
Low background
Straightforward miniaturization from 96- or 384-well microplates to high density assay formats such as 384-well low volume and 1536-well plates
Stable signal, providing flexibility in time of readout or size of assays

How it works

Phospho-AKT (Ser473) assay principle

The Phospho-AKT (Ser473) assay measures AKT when phosphorylated at Ser473. Contrary to Western Blot, the assay is entirely plate-based and does not require gels, electrophoresis or transfer. The Phospho-AKT (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 proteins phosphorylation state under a no-wash assay format.

Phospho-AKT (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-AKT (Ser473) assay 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 (Ser473) assay 1-plate assay protocol

Detection of Phosphorylated AKT (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.

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

Assay validation

Phospho-AKT (Ser473) Assay IGF Dose-Response on HEK 293 cells

Results obtained on HEK cells plated using 3 cell densities (12,500 - 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 was measured after 3.5 hours using the two-plate assay protocol.

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.

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.

phospho-pathway-akt-64akspeg-64akspeh-64akspet-64akspey3.svg

Specifications

Other specifications

Application	Cell Signaling
Brand	HTRF
Detection Modality	HTRF
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
Unit Size	500 assay points