Akt, Phosphorylated (S473), BioAssay™ ELISA 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.
For the quantitative determination of Akt phosphorylated at S473 in cell lysates.
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.
Principle of the Assay:
The Akt, phosphorylated (S473) ELISA kit 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.
A1124-95A; Microtiter Plate, 12 strips x 8wells. Coated with Phospho-Akt (S473) pan-specific antibody. Return unused wells to the foil pouch containing the desiccant pack, reseal
along entire edge of zip-seal. May be stored for up to 1 month at 2-8° C.
A1124-95B: Standard (Phospo-Akt (S473), Pan-specific), 2x 185ng. Each vial vial contains lyophilized recombinant phospho-Akt2 in a buffered protein base with preservatives. Use within 1 hour of reconstitution. Use a fresh standard for each assay.
A1124-95C: Detection Antibody, 1x1vial. Contains 7.5ug of lyophilized biotinylated mouse anti-phospho-Akt (S473) pan-specific antibody.
A1124-95D: Lysis Buffer, 1x21ml. Cell lysing buffer with phosphatase inhibitors and preservatives.
A1124-95E: Sample Diluent Concentrate (5X), 1x21 ml. 5-fold concentrated buffer with preservatives.
A1124-95F: Reagent Diluent Concentrate (10X), 1x21ml. 10-fold concentrated solution of buffered protein base with preservatives.
A1124-95G: Wash Buffer Concentrate (25X), 1x21ml. 25-fold concentrated solution of buffered yellow surfactant with preservative. May turn yellow over time.
A1124-95H: Color Reagent A, 1x12.5ml. Stabilized hydrogen peroxide.
A1124-95I: Color Reagent B, 1x12.5ml Stabilized chromogen (tetramethylbenzidine).
A1124-95J: Streptavidin-HRP, 1x1ml. Streptavidin conjugated to horseradish peroxidase.
A1124-95K: Stop Solution, 1x6ml. 2N sulfuric acid.
Storage and Stability:
Store all kit components at 4°C.
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.