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Typical phospho-Akt (Thr308) Standard Curve 100 µL of progressive 2 fold dilutions of the phospho-Akt (Thr308) standard included in the kit and...
phospho-Akt (Thr308) STAR ELISA Kit
Description:
phospho-Akt (Thr308) STAR ELISA Kit
Trade Name:
- STAR ELISA
- Upstate (Millipore)
Qty/Pk:
96 assays
Background Information:
The UPSTATE colorimetric STAR (Signal Transduction Assay Reaction) ELISA kit is a solid phase sandwich enzyme linked immunosorbent assay that provides a fast, sensitive method to detect specific levels of signaling targets in whole cell extracts. The AKT plate is coated with a specific mouse monoclonal anti-AKT capture antibody on the microwells of the 96-well clear plate. Sample lysate or the standard included in the kit are incubated in the microwells allowing AKT antigen to be captured in the plate wells. The plate is then washed to remove any non-bound unspecific material. The wells are then incubated with a specific rabbit anti-AKT antibody to detect the captured phospho-AKT (Thr308) on the plate well. The unbound detection antibody is washed away followed by incubation with an HRP-conjugated anti-rabbit antibody. This allows for a sensitive enzymatic detection of the sample. After the addition of TMB substrate and stop solution the absorbance is measured at 450 nm using a plate reader. The entire assay takes less than 5 hours to complete with minimal hands-on time. Many of the reagents are supplied in ready-to use formulations for ease of use. The kit also includes a standard that is run as both a positive control and to develop a standard curve.
II. Akt BACKGROUND
Akt (Protein Kinase B), a Ser/Thr kinase, is a major known effecter of the PI3 Kinase pathway and is involved in multiple signaling pathways that relate to many biological processes including glucose metabolism, cell survival/apoptosis, cell cycle control, angiogenesis, differentiation, and cell growth and proliferation. Akt is activated by ligand-stimulated growth factor receptor signaling that activates the Phosphatidylinositol 3-kinase (PI3 Kinase, PI3K) dependent manner. PKB is one of the most frequently hyperactivated protein kinases in human cancers. In mammals three isoforms of Akt (Akt1/PKBα, Akt2/PKBβ, and Akt3/PKBγ) exists. They exhibit a high degree of homology, but differ slightly in the localization of their regulatory phosphorylation sites. Akt1 is the predominant isoform that is in most tissues and is thought to have a dominant role in growth, survival, embryonic development, and post-natal survival. Additionally, Akt1/PKBα is required for adipocyte differentiation, where as Akt2/PKBβ and Akt3/PKBγ are not. Akt2 is strongly correlated with the regulation of glucose homeostasis and is the predominant PKB isoform expressed in insulin-responsive tissues where defective Akt2 results in impaired insulin-stimulated glucose uptake in muscle and adipocytes. Akt3 is abundant in brain tissue. Each Akt isoform is composed of three functionally distinct regions: an N-terminal Pleckstrin Homology (PH) domain that provides a lipid-binding module to direct Akt to PIP3 at the cell membrane as a result of PI3 Kinase (PI3K) activity that is necessary for its activation, a central catalytic domain, and a C-terminal hydrophobic motif. The activation and regulation of AKT is dependent on a dual regulatory mechanism that requires both its translocation to the plasma membrane and dual phosphorylation on Thr308 and Ser473 by PDK1 and the TORC2 complex, respectively. This is accomplished by the generation and build-up of PIP3 by PI3K in conjunction with reduced PTEN function that results in the activation of PDK1 (3-phosphoinositide-dependent protein kinase-1) and the recruitment of AKT to the plasma membrane by direct interaction with its PH domain. The activated PDK1 then in turn phosphorylates Akt on Thr308 in its activation loop. This phosphorylation is necessary and sufficient for AKT activation; however maximal activation requires the additional phosphorylation at Ser473. Another kinase complex, recently identified as TORC2, which is composed of the mTOR, Rictor, GL, Sin1, and Protor1 and 2 (previously referred to as the unidentified kinase PDK2), phosphorylates AKT on Ser473 in its hydrophobic motif. After Akt is activated, it is liberated from the plasma membrane and released into the cytosol and nucleus where it interacts with and phosphorylates multiple binding partners. It has been shown to phosphorylate over 40 substrates, some of which are activated by phosphorylation such as mTOR, AS160, PRAS40 (Thr246), IKK, MDM2, NFκB, and TSC1&2 and some that are inhibited by its phosphorylation that include Bad (Ser136), GSK3 (Ser9), FKHR (Ser256), and Caspase 9 (Ser196).
Just as these two AKT phosphorylation sites are phosphorylated by two separate mechanisms, they are both regulated by two different phosphatases. The dephosphorylation and subsequent inactivation of AKT is much less understood than its activation. It was not until the recent discovery of two new phosphatases, PHLPP1 and PHLPP2 (PH domain leucine-rich repeat protein phosphatase) that the process was better elucidated. Dephosphorylation of AKT at Ser473, but not at Thr308, was found to be mediated by one or both of the PHLPP family of phosphatases. Another more promiscuous phosphatase, PP2A, is now believed to dephosphorylate AKT on the PDK1 phosphorylation site at Thr308. Together these phosphatases help regulate the activity of AKT. With AKT having so many signaling partners and its involvement in multiple signaling pathways and cellular mechanisms, it is no wonder why AKT is so well studied and a highly sought after drug target.
II. Akt BACKGROUND
Akt (Protein Kinase B), a Ser/Thr kinase, is a major known effecter of the PI3 Kinase pathway and is involved in multiple signaling pathways that relate to many biological processes including glucose metabolism, cell survival/apoptosis, cell cycle control, angiogenesis, differentiation, and cell growth and proliferation. Akt is activated by ligand-stimulated growth factor receptor signaling that activates the Phosphatidylinositol 3-kinase (PI3 Kinase, PI3K) dependent manner. PKB is one of the most frequently hyperactivated protein kinases in human cancers. In mammals three isoforms of Akt (Akt1/PKBα, Akt2/PKBβ, and Akt3/PKBγ) exists. They exhibit a high degree of homology, but differ slightly in the localization of their regulatory phosphorylation sites. Akt1 is the predominant isoform that is in most tissues and is thought to have a dominant role in growth, survival, embryonic development, and post-natal survival. Additionally, Akt1/PKBα is required for adipocyte differentiation, where as Akt2/PKBβ and Akt3/PKBγ are not. Akt2 is strongly correlated with the regulation of glucose homeostasis and is the predominant PKB isoform expressed in insulin-responsive tissues where defective Akt2 results in impaired insulin-stimulated glucose uptake in muscle and adipocytes. Akt3 is abundant in brain tissue. Each Akt isoform is composed of three functionally distinct regions: an N-terminal Pleckstrin Homology (PH) domain that provides a lipid-binding module to direct Akt to PIP3 at the cell membrane as a result of PI3 Kinase (PI3K) activity that is necessary for its activation, a central catalytic domain, and a C-terminal hydrophobic motif. The activation and regulation of AKT is dependent on a dual regulatory mechanism that requires both its translocation to the plasma membrane and dual phosphorylation on Thr308 and Ser473 by PDK1 and the TORC2 complex, respectively. This is accomplished by the generation and build-up of PIP3 by PI3K in conjunction with reduced PTEN function that results in the activation of PDK1 (3-phosphoinositide-dependent protein kinase-1) and the recruitment of AKT to the plasma membrane by direct interaction with its PH domain. The activated PDK1 then in turn phosphorylates Akt on Thr308 in its activation loop. This phosphorylation is necessary and sufficient for AKT activation; however maximal activation requires the additional phosphorylation at Ser473. Another kinase complex, recently identified as TORC2, which is composed of the mTOR, Rictor, GL, Sin1, and Protor1 and 2 (previously referred to as the unidentified kinase PDK2), phosphorylates AKT on Ser473 in its hydrophobic motif. After Akt is activated, it is liberated from the plasma membrane and released into the cytosol and nucleus where it interacts with and phosphorylates multiple binding partners. It has been shown to phosphorylate over 40 substrates, some of which are activated by phosphorylation such as mTOR, AS160, PRAS40 (Thr246), IKK, MDM2, NFκB, and TSC1&2 and some that are inhibited by its phosphorylation that include Bad (Ser136), GSK3 (Ser9), FKHR (Ser256), and Caspase 9 (Ser196).
Just as these two AKT phosphorylation sites are phosphorylated by two separate mechanisms, they are both regulated by two different phosphatases. The dephosphorylation and subsequent inactivation of AKT is much less understood than its activation. It was not until the recent discovery of two new phosphatases, PHLPP1 and PHLPP2 (PH domain leucine-rich repeat protein phosphatase) that the process was better elucidated. Dephosphorylation of AKT at Ser473, but not at Thr308, was found to be mediated by one or both of the PHLPP family of phosphatases. Another more promiscuous phosphatase, PP2A, is now believed to dephosphorylate AKT on the PDK1 phosphorylation site at Thr308. Together these phosphatases help regulate the activity of AKT. With AKT having so many signaling partners and its involvement in multiple signaling pathways and cellular mechanisms, it is no wonder why AKT is so well studied and a highly sought after drug target.
Key Applications:
ELISA
Species Reactivity:
- Human
- Mouse
- Rat
Usage Statement:
Unless otherwise stated in our catalog or other company documentation accompanying the product(s), our products are intended for research use only and are not to be used for any other purpose, which includes but is not limited to, unauthorized commercial uses, in vitro diagnostic uses, ex vivo or in vivo therapeutic uses or any type of consumption or application to humans or animals.
Entrez Gene Summary:
The serine-threonine protein kinase encoded by the AKT1 gene is catalytically inactive in serum-starved primary and immortalized fibroblasts. AKT1 and the related AKT2 are activated by platelet-derived growth factor. The activation is rapid and specific, and it is abrogated by mutations in the pleckstrin homology domain of AKT1. It was shown that the activation occurs through phosphatidylinositol 3-kinase. In the developing nervous system AKT is a critical mediator of growth factor-induced neuronal survival. Survival factors can suppress apoptosis in a transcription-independent manner by activating the serine/threonine kinase AKT1, which then phosphorylates and inactivates components of the apoptotic machinery. Multiple alternatively spliced transcript variants have been found for this gene.
Brand Family:
Upstate
Sensitivity:
Sensitivity: 1 unit/mL.
Range of Detection: 1.6 to 100 units/mL
Range of Detection: 1.6 to 100 units/mL
Storage Conditions:
1 year at 4°C
Modifications:
Phosphorylation
Gene Symbol:
- AKT1
- RAC
- PRKBA
- MGC99656
- RAC-ALPHA
- RAC-PK-alpha
- C-AKT
- PKB
- AKT
Product Name:
phospho-Akt (Thr308) STAR ELISA Kit
Alternate Names:
PKB
Materials Required but Not Delivered:
1. Multi-channel or repeating pipettes
2. Plate shaker (optional)
3. Pipettors & tips capable of accurately measuring 1-1000 %micro;L
4. Graduated serological pipettes
5. 96-well microtiter Plate Reader with 450 nm filter
6. Graphing software for plotting data or graph paper for manual plotting of data
7. Microfuge tubes for standard and sample dilutions
8. Mechanical vortex
9. 1 liter container
10. Distilled or deionized water
2. Plate shaker (optional)
3. Pipettors & tips capable of accurately measuring 1-1000 %micro;L
4. Graduated serological pipettes
5. 96-well microtiter Plate Reader with 450 nm filter
6. Graphing software for plotting data or graph paper for manual plotting of data
7. Microfuge tubes for standard and sample dilutions
8. Mechanical vortex
9. 1 liter container
10. Distilled or deionized water
Analytes Available:
Akt (PKB)
Quality Assurance:
Routinely evaluated by ELISA.
Components:
-
1. Capture Plate pre-coated with anti-AKT antibody: (Part No. 17-456A) One pre-coated 96-stripwell immunoplate sealed in a foil pouch.
-
2. Anti-phospho-Akt (Thr308) detection antibody: (Part No. 17-456B) One bottle (11 mL) of anti- phospho-Akt (Thr308) detection antibody containing sodium azide, ready to use.
-
3. ELISA Diluent: (Part No. 17-456C) One bottle (25 mL) of ELISA Diluent containing sodium azide, ready to use.
-
4. 25X ELISA Wash Buffer: (Part No. 17-456D) One bottle (50 mL) of 25X ELISA Wash Buffer.
-
5. Anti-Rabbit IgG HRP conjugate: (Part No. 17-456E) One vial (125 µL) of 100X anti-rabbit HRP conjugate containing thimerosol.
-
6. HRP Diluent: (Part No. 17-456F) One bottle (25 mL) of HRP Diluent containing thimerosol.
-
7. TMB Solution: (Part No. 17-456G) One bottle (25 mL) of stabilized tetramethylbenzidine (TMB), ready to use.
-
8. Stop Solution: (Part No. 17-456H) One bottle (25 mL) of stop solution, ready to use.
-
9. Phospho-Akt (Thr308) Standard: (Part No. 17-456I) Four vials of phospho-Akt (Thr308) standard, lyophilized.
- 10. Plate Covers: Two plate covers.
UniProt Number:
Detection Methods:
- Chromogenic
- Chromogenic
Entrez Gene Number: