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HTRF Human Total AKT2 Detection Kit, 500 Assay Points

This HTRF kit is designed to monitor the expression level of cellular AKT2 and can be used as a normalization assay for the phospho-AKT2 kit.

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 to monitor the expression level of cellular AKT2 and can be used as a normalization assay for the phospho-AKT2 kit.

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Product Variants

Unit Size: 500 Assay Points

Part #:

63ADK081PEG

List Price

USD 2,147.00

Unit Size: 10,000 Assay Points

Part #:

63ADK081PEH

List Price

USD 12,490.00

Product information

Overview

This kit is designed to monitor the modulation in expression of total AKT2, and is used as a normalization assay with our Phospho-AKT2 (Ser473) kit. 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. AKT2 in particular plays a critical role in the insulin pathway, and thus has important applications in diabetes and metabolic research.

Specifications

Other Specifications

Application	Cell Signaling
Brand	HTRF
Detection Modality	HTRF
Lysis Buffer Compatibility	Lysis Buffer 1
Molecular Modification	Total
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

How it works

Total-AKT2 assay principle

The Total-AKT2 assay quantifies the expression level of AKT2 in a cell lysate. Contrary to Western Blot, the assay is entirely plate-based and does not require gels, electrophoresis or transfer. The Total-AKT2 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 AKT2 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-AKT2 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 AKT2 HTRF detection reagents. This protocol enables the cells' viability and confluence to be monitored.

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Total-AKT2 1-plate assay protocol

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

Assay validation

Total AKT2 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 AKT2 was added before adding 4 µL of the HTRF total AKT2 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.