		Technical Data
A1124-95	Akt, Phosphorylated (S473), BioAssay™ ELISA Kit	1 Kit

Kits and Assays

Storage: 4°CShipping: Blue Ice

The serine/threonine kinase Akt, also known as protein kinase B (PKB), is a central player in such diverse cellular processes as glucose uptake, cell cycle progression, and apoptosis. In mammals, three highly homologous members define the Akt family: Akt1 (PKBa), Akt2 (PKBb), and Akt3 (PKBg). Akt1 is the most ubiquitously expressed member, while Akt2 is expressed predominantly in insulin target tissues such as liver, skeletal muscle, and fat. Akt3 is expressed most selectively, in brain and testis. All three Akts share a conserved structure: an amino-terminal pleckstrin homology domain to interact with membrane phosphoinositides; a central kinase domain; and a carboxyl-terminal regulatory domain that contains the hydrophobic motif, a hallmark of members of the AGC kinase superfamily. Akt signaling is activated by receptor tyrosine kinases, B- and T-cell receptors, cytokine receptors, and other mechanisms that activate phosphoinositide 3-kinase (PI 3-K). Active PI 3-K generates the second messenger phosphatidylinositol-3,4,5-trisphosphate (PIP3), which recruits Akt to the plasma membrane via the pleckstrin homology domain for subsequent phosphorylation. Phosphoinositide-dependent kinase-1 (PDK-1) phosphorylates Akt in the activation loop of the kinase domain (T308 of Akt1) and an unknown kinase, possibly the rictor-mTOR complex, phosphorylates Akt in the hydrophobic motif (S473 of Akt1) of the regulatory domain. Phosphorylation of both sites is necessary for full Akt activation. Downstream Akt substrates include regulators of metabolism and apoptosis, implicating Akt1-3 as targets for diabetes and cancer therapies.

Sample Type:
Cell lysates

Intended Use:
For the quantitative determination of Akt phosphorylated at S473 in cell lysates.

Specificity:
Recognizes Akt family members phosphorylated at sites corresponding to S473 of Akt1 from human, mouse, and rat. Specificity was demonstrated using both peptide competition and cross-reactivity analysis.

Test Principle:
This Surveyor IC Immunoassay employs a two-site sandwich ELISA to quantitate Akt phosphorylated at S473 in cell lysates. An antibody specific for Akt1, Akt2, and Akt3, binding both phosphorylated and unphosphorylated protein, has been pre-coated onto a microplate. Standards and samples are added and Akt1, Akt2, and Akt3 present are bound by the immobilized antibody. After washing away unbound material, a biotinylated detection antibody recognizing Akt1 phosphorylated at S473, Akt2 phosphorylated at S474, and Akt3 phosphorylated at S472 is used to detect only phosphorylated protein utilizing a standard streptavidin-HRP format. Substrate Solution is added to the wells and color develops in proportion to the amount of phosphorylated Akt bound in the initial step. The color development is stopped and the intensity of the color is measured.

Kit Components:
Phospho-Akt (S473), Pan Specific Microplate: 12 strips x 8 wells
Phospho-Akt (S473), Pan Specific Standard: 1x245ng
Phospho-Akt (S473), Pan Specific Detection Antibody: 1x30ug
Lysis Buffer 6: 1x21ml
Sample Diluent Concentrate 1 (5X): 1x21ml
Reagent Diluent Concentrate 2 (10X): 1x21ml
Wash Buffer Concentrate (25X): 1x21ml
Color Reagent A: 1x12.5ml
Color Reagent B: 1x12.5ml
Streptavidin conjugated to HRP: 1x1ml
Stop Solution: 1x6ml
Plate Covers: 4 adhesive strips

Storage and Stability:
See Kit Protocol for detailed storage instructions.

Important Note: This product as supplied is intended for research use only, not for use in human, therapeutic or diagnostic applications without the expressed written authorization of United States Biological.

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Intended for research use only. Not for use in human, therapeutic, or diagnostic applications.