This HTRF kit is designed for robust cell-based quantification of AKT2 modulation, phosphorylated on Ser473.
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).
The Phospho-AKT2 (Ser473) kit is designed for the robust quantification of AKT2 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. AKT2 in particular plays a critical role in the insulin pathway, and thus has important applications in diabetes and metabolic research.
Assay Points |
500
|
---|---|
Assay Technology |
HTRF
|
Brand |
HTRF
|
Product Group |
Kit
|
Quantity |
1
|
Shipping Conditions |
Shipped in Dry Ice
|
Therapeutic Area |
Metabolism/Diabetes
NASH/Fibrosis
Oncology & Inflammation
|
Unit Size |
500 Assay Points
|
The Phospho-AKT2 (Ser473) assay measures AKT2 when phosphorylated at Ser473. Contrary to Western Blot, the assay is entirely plate-based and does not require gels, electrophoresis or transfer. The Phospho-AKT2 (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.
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-AKT2 (Ser473) HTRF detection reagents. This protocol enables the cells' viability and confluence to be monitored.
Detection of Phosphorylated AKT2 (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.
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 or was added before adding 4 µL of the HTRF phospho-AKT2 (Ser473) detection reagents. The HTRF signal was recorded after an overnight incubation at room temperature.
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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