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HTRF Human Total AKT2 Detection Kit, 500 Assay Points
HTRF Human Total AKT2 Detection Kit, 500 Assay Points
PHOTO TOTAL AKT 2 (
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).
| Feature | Specification |
|---|---|
| Application | Cell Signaling |
| Sample Volume | 16 µL |
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).
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
HTRF Human Total AKT2 Detection Kit, 500 Assay Points
PHOTO TOTAL AKT 2 (
HTRF Human Total AKT2 Detection Kit, 500 Assay Points
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
| 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
|
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HTRF Human Total AKT2 Detection Kit, 500 Assay Points
HTRF Human Total AKT2 Detection Kit, 500 Assay Points
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.
Total-AKT2 1-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.
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.
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