Key features and details
- Assay type: Cell-based (quantitative)
- Detection method: Luminescent
- Sample type: Adherent cells, Suspension cells
- Reacts with: Mouse, Rat, Human
Product namePhospho-AKT (S473) + Total In-Cell ELISA Kit (Chemiluminescent)
See all AKT1 kits
Sample typeAdherent cells, Suspension cells
Assay typeCell-based (quantitative)
Assay durationMultiple steps standard assay
Species reactivityReacts with: Mouse, Rat, Human
Phospho-AKT (S473) + Total In-Cell ELISA (Chemiluminescent) (ab207452) provides a simple, efficient, cell-based method to monitor proteins activated by phosphorylation. The kit is designed specifically to quantify activated (phosphorylated) AKT and/or total AKT. Cells are cultured in 96-well plates and stimulated to induce the pathway of interest. Following stimulation, the cells are rapidly fixed to preserve activation-specific protein modifications. Each well is then incubated with a primary antibody that recognizes either phosphorylated AKT or total AKT. Subsequent incubation with secondary HRP-conjugated antibody and developing solution provides an easily quantified chemiluminescent readout. The relative number of cells in each well is then determined using the provided Crystal Violet solution. The 96-well plate format is suitable for high-throughput screening applications.
The Phospho-AKT (S473) + Total In-Cell ELISA Kit (Chemiluminescent) contains 96-well plates and two primary antibodies. The phospho-AKT antibody was raised in rabbit against a synthetic phospho-peptide corresponding to the sequence surrounding Ser473 of mouse AKT. This antibody recognizes only phosphorylated AKT. The total-AKT antibody was raised in rabbit and recognizes AKT regardless of its phosphorylation state. The kit can be used to study phosphorylated AKT relative to cell number or to determine AKT phosphorylation relative to the total AKT protein found in the cells. Once the phospho-AKT and total-AKT signals have been normalized for cell number, a comparison of the ratio of phosphorylated AKT to total AKT for each of the cell growth conditions can be made. The provided total-AKT antibody can be used as a positive control to demonstrate that the cells contain AKT, the kit reagents are functional and that the protocol is performed correctly. Also, because fixed cells are stable for several weeks, many plates can be prepared simultaneously and then the assay performed when desired.
This kit be used to study phosphorylated AKT relative to cell number or be used to determine AKT phosphorylation relative to the total AKT protein found in the cells. Once the phospho-AKT and total-AKT signals have been normalized for cell number, a comparison of the ratio of phosphorylated AKT to total AKT for each of the cell growth conditions can be made.
The serine/threonine protein kinase AKT is a key player in the regulation of cell survival. AKT functions to promote cell survival by inhibiting apoptosis through its ability to phosphorylate and inactivate several targets including Bad, caspase-9 and Forkhead transcription factor. AKT also plays an important role in the pathologies of degenerative diseases and cancer. The PI-3 kinase/AKT signaling pathway is directly linked to the malignant progression of gastric tumor cells, and activation of AKT may be an early marker for tumor progression in melanoma. Interestingly, sex-related differences in the levels of phospho-AKT have also been reported.
AKT is downstream of many growth factor signaling cascades16, 17, including EGF, IGF-I and heregulin. In particular, in the hormone-dependent breast cancer cell line MCF-7, the mitogenic growth factors EGF and IGF-I have been shown to regulate estrogen receptor-alpha gene expression via AKT. Estradiol treatment can also rapidly activate AKT.
Upstream kinases are involved in the activation of AKT. Stimulation of phosphatidylinositol-3 kinase (PI-3K) leads to the generation of phosphatidylinositol triphosphate, which activates PDK1 (phosphoinositide-dependent protein kinase 1). Activated PDK1 then phosphorylates AKT on threonine 308 (Thr308), which is followed by autophosphorylation of AKT on serine 473 (Ser473). Such phosphorylation is essential for the regulation of AKT and for the maintenance of its activity. Furthermore, binding of PI-3K-generated phospholipids to AKT is critical to its activation and translocation from the cytoplasm to the plasma membrane. PTEN (phosphatase and tensin homolog deleted on chromosome ten) has been shown to be a major negative regulator of the PI-3K/AKT signaling pathway.
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Storage instructionsPlease refer to protocols.
Components 1 x 96 tests 5 x 96 tests 1% SDS Solution 1 x 22ml 5 x 22ml 10% Triton X-100 1 x 10ml 5 x 10ml 10X PBS 1 x 120ml 5 x 120ml 1X Antibody Blocking Buffer 1 x 22ml 5 x 22ml 1X Antibody Dilution Buffer 1 x 30ml 5 x 30ml 96-well tissue culture plate 2 units 10 units Anti-rabbit HRP-conjugated IgG 1 x 11µl 5 x 11µl Chemiluminescent Reagent 2 x 2ml 10 x 2ml Crystal Violet Solution 1 x 22ml 5 x 22ml Phospho-AKT antibody 1 x 18µl 5 x 18µl Plate sealer 2 units 10 units Reaction Buffer 2 x 4ml 10 x 4ml Total-AKT antibody 1 x 9µl 5 x 9µl
FunctionPlays a role as a key modulator of the AKT-mTOR signaling pathway controlling the tempo of the process of newborn neurons integration during adult neurogenesis, including correct neuron positioning, dendritic development and synapse formation (By similarity). General protein kinase capable of phosphorylating several known proteins. Phosphorylates TBC1D4. Signals downstream of phosphatidylinositol 3-kinase (PI(3)K) to mediate the effects of various growth factors such as platelet-derived growth factor (PDGF), epidermal growth factor (EGF), insulin and insulin-like growth factor I (IGF-I). Plays a role in glucose transport by mediating insulin-induced translocation of the GLUT4 glucose transporter to the cell surface. Mediates the antiapoptotic effects of IGF-I. Mediates insulin-stimulated protein synthesis by phosphorylating TSC2 at 'Ser-939' and 'Thr-1462', thereby activating mTORC1 signaling and leading to both phosphorylation of 4E-BP1 and in activation of RPS6KB1. Promotes glycogen synthesis by mediating the insulin-induced activation of glycogen synthase. The activated form can suppress FoxO gene transcription and promote cell cycle progression. Essential for the SPATA13-mediated regulation of cell migration and adhesion assembly and disassembly.
Tissue specificityExpressed in all human cell types so far analyzed. The Tyr-176 phosphorylated form shows a significant increase in expression in breast cancers during the progressive stages i.e. normal to hyperplasia (ADH), ductal carcinoma in situ (DCIS), invasive ductal carcinoma (IDC) and lymph node metastatic (LNMM) stages.
Involvement in diseaseDefects in AKT1 are a cause of susceptibility to breast cancer (BC) [MIM:114480]. A common malignancy originating from breast epithelial tissue. Breast neoplasms can be distinguished by their histologic pattern. Invasive ductal carcinoma is by far the most common type. Breast cancer is etiologically and genetically heterogeneous. Important genetic factors have been indicated by familial occurrence and bilateral involvement. Mutations at more than one locus can be involved in different families or even in the same case.
Defects in AKT1 are associated with colorectal cancer (CRC) [MIM:114500].
Defects in AKT1 are associated with susceptibility to ovarian cancer [MIM:604370]; also called susceptibility to familial breast-ovarian cancer type 1 (BROVCA1).
Sequence similaritiesBelongs to the protein kinase superfamily. AGC Ser/Thr protein kinase family. RAC subfamily.
Contains 1 AGC-kinase C-terminal domain.
Contains 1 PH domain.
Contains 1 protein kinase domain.
DomainBinding of the PH domain to the phosphatidylinositol 3-kinase alpha (PI(3)K) results in its targeting to the plasma membrane. The PH domain mediates interaction with TNK2 and Tyr-176 is also essential for this interaction.
The AGC-kinase C-terminal mediates interaction with THEM4.
modificationsPhosphorylation on Thr-308, Ser-473 and Tyr-474 is required for full activity. Activated TNK2 phosphorylates it on Tyr-176 resulting in its binding to the anionic plasma membrane phospholipid PA. This phosphorylated form localizes to the cell membrane, where it is targeted by PDPK1 and PDPK2 for further phosphorylations on Thr-308 and Ser-473 leading to its activation. Ser-473 phosphorylation by mTORC2 favors Thr-308 phosphorylation by PDPK1. Ser-473 phosphorylation is enhanced by interaction with AGAP2 isoform 2 (PIKE-A). Ser-473 phosphorylation is enhanced in focal cortical dysplasias with Taylor-type balloon cells.
Ubiquitinated; undergoes both 'Lys-48'- and 'Lys-63'-linked polyubiquitination. TRAF6-induced 'Lys-63'-linked AKT1 ubiquitination is critical for phosphorylation and activation. When ubiquitinated, it translocates to the plasma membrane, where it becomes phosphorylated. When fully phosphorylated and translocated into the nucleus, undergoes 'Lys-48'-polyubiquitination catalyzed by TTC3, leading to its degradation by the proteasome.
Cellular localizationCytoplasm. Nucleus. Cell membrane. Nucleus after activation by integrin-linked protein kinase 1 (ILK1). Nuclear translocation is enhanced by interaction with TCL1A. Phosphorylation on Tyr-176 by TNK2 results in its localization to the cell membrane where it is targeted for further phosphorylations on Thr-308 and Ser-473 leading to its activation and the activated form translocates to the nucleus.
- Information by UniProt
- AKT 1
ab207452 has been referenced in 1 publication.
- Li J et al. Nitric Oxide Synthase Is Involved in Follicular Development via the PI3K/AKT/FoxO3a Pathway in Neonatal and Immature Rats. Animals (Basel) 10:N/A (2020). PubMed: 32033275