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HTRF Human Phospho-AKT3 (Ser473) Detection Kit, 500 Assay Points

This HTRF kit is designed for cell-based quantification of AKT3 expression, phosphorylated on Ser473, as a readout of Pi3K pathway activation.

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

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This HTRF kit is designed for cell-based quantification of AKT3 expression, phosphorylated on Ser473, as a readout of Pi3K pathway activation.

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Product Variants

Unit Size: 500 Assay Points

Part #:

63ADK082PEG

List Price

USD 2,147.00

Unit Size: 10,000 Assay Points

Part #:

63ADK082PEH

List Price

USD 12,490.00

Product information

Overview

The Phospho-AKT3 (Ser473) kit is designed for the robust quantification of AKT3 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. AKT3 in particular plays a critical role in neuroscience research.

Specifications

Other Specifications

Application	Cell Signaling
Brand	HTRF
Detection Modality	HTRF
Lysis Buffer Compatibility	Lysis Buffer 1
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	Neuroscience
Unit Size	500 Assay Points

Video gallery

How it works

Phospho-AKT3 (Ser473) assay principle

The Phospho-AKT3 (Ser473) assay measures AKT3 when phosphorylated at Ser473. Contrary to Western Blot, the assay is entirely plate-based and does not require gels, electrophoresis or transfer. The Phospho-AKT3 (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-AKT3 (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-AKT3 (Ser473) 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-AKT3 (Ser473)1-plate assay protocol

Detection of Phosphorylated AKT3 (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 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 phospho-AKT3 (Ser473) detection reagents. The HTRF signal was recorded after an overnight incubation at room temperature.

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.