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HTRF Human Phospho-AKT1 (Ser473) Detection Kit, 500 Assay Points

This HTRF kit is designed to monitor AKT1 phosphorylation 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 & disposal requirements under European REACH regulations (EC 1907/2006).
Product Variants
Part number: 63ADK078PEG
Unit Size: 500 Assay Points
List price: USD 2,147.00
Your price:
USD 2,147.00
USD 2,147.00 /each
Part number: 63ADK078PEH
Unit Size: 10,000 Assay Points
List price: USD 12,490.00
Your price:
USD 0.00
USD 12,490.00 /each

Overview

The phospho-AKT1 (Ser473) kit is designed for robust quantification of AKT1 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. AKT1 in particular plays a critical role in cell survival, and thus has important applications in cancer research.

Specifications

Assay Points
500
Assay Target Type
Kit
Assay Technology
HTRF
Brand
HTRF
Quantity
1
Shipping Conditions
Shipped in Dry Ice
Therapeutic Area
Metabolism/Diabetes
NASH/Fibrosis
Unit Size
500 Assay Points

Video gallery

How it works

Phospho-AKT1 (Ser473) assay principle

The Phospho-AKT1 (Ser473) assay measures AKT1 when phosphorylated at Ser473. Contrary to Western Blot, the assay is entirely plate-based and does not require gels, electrophoresis or transfer. The Phospho-AKT1 (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-how-it-works-phospho-btk-tyr223-a1.svg

 

Phospho-AKT1 (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-AKT1 (Ser473) HTRF detection reagents. This protocol enables the cells' viability and confluence to be monitored.

Two-plate protocol of the HTRF total BRD2 assay

 

Phospho-AKT1 (Ser473) 1-plate assay protocol

Detection of Phosphorylated AKT1 (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.

One-plate protocol of the HTRF total BRD2 assay

 

Assay validation

Phospho AKT1 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 small volume white microplates and 2 µL of different concentration of blocking peptide specific for AKT1 was added before adding 4 µL of the HTRF phospho AKT1 (Ser473) detection reagents. The HTRF signal was recorded after an overnight incubation at room temperature.

1assay-validation-akt1-phospho-s473-1.svg

Simplified pathway

Phospho AKT1 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 small volume white microplates and 2 µL of different concentration of blocking peptide specific for AKT1 was added before adding 4 µL of the HTRF phospho AKT1 (Ser473) detection reagents. The HTRF signal was recorded after an overnight incubation at room temperature.

2phospho-pathway-akt-63adk078peg-63adk078peh-63adk078pey.svg

 

Resources

1-4 of 4 Resources
Guide
HTRF solutions, guide to major applications

This guide provides you an overview of HTRF applications in several therapeutic areas.

Guide
Protein degradation awareness in a single guide

<p>An in-depth review of molecular and cellular pathways</p><p>The maintenance of proteostasis, the biological mechanisms that control the...</p>

Guide
Solutions for in-depth studies of diabetes

<p>A comprehensive listing of Type II diabetes pathways</p><p>Type II diabetes disease cases are constantly increasing, especially for...</p>

Application Note Icon Image
Application Note
TCR phospho signaling investigation with HTRF

Study your pathway of interest in PBMC and T cells

Combine models with a technique that maximizes each one’s relevance: this note...

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SDS, COAs, Manuals and more

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