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AKT 1/2/3 Total ELISA Kit
Intra-assay Sample n Mean SD CV% Overall 6 1.8% Inter-assay Sample n Mean SD CV% Overall 3 9.5%
< 1 µg/ml
0.05 % - 3.12 %
One step assay
Reacts with: Mouse, Human
Predicted to work with: Rat
AKT 1/2/3 Total (ab253298) is a single-wash 90 min sandwich ELISA designed for the semi-quantitative measurement oftotal AKT 1/2/3 protein in human and mouse celllysates. It uses our proprietary SimpleStep ELISA® technology. Semi-quantitate total AKT 1/2/3 with 1 µg/mLsensitivity.
SimpleStep ELISA® technology employs capture antibodies conjugated to an affinity tag that is recognized by the monoclonal antibody used to coat our SimpleStep ELISA® plates. This approach to sandwich ELISA allows the formation of the antibody-analyte sandwich complex in a single step, significantly reducing assay time. See the SimpleStep ELISA® protocol summary in the image section for further details. Our SimpleStep ELISA® technology provides several benefits:
-Single-wash protocol reduces assay time to 90 minutes or less
-High sensitivity, specificity and reproducibility from superior antibodies
-Fully validated in biological samples
-96-wells plate breakable into 12 x 8 wells strips
A 384-well SimpleStep ELISA® microplate (ab203359) is available to use as an alternative to the 96-well microplate provided with SimpeStep ELISA® kits.
AKT isoforms belong to a conserved family of serine/threonine protein kinases. AKT plays a central role in cell survival and inhibiting apoptosis, by phosphorylating and inactivating several targets. AKT is activated by insulin and various other agonists, which promote transport to the plasma membrane, where the activation loop of AKT is phosphorylated at Thr308 by PDK1. The carboxy terminus is phosphorylated at Ser473 by mammalian target of rapamycin complex 2 (TORC2).
AKT substrates include Bad, forkhead transcription factors, and caspase-9. Another vital AKT function is the regulation of glycogen synthesis through phosphorylation of GSK-3. AKT phosphorylation also prevents GSK-3β mediated phosphorylation and degradation of cyclin D1, and negatively regulates the cyclin dependent kinase inhibitors p27 Kip and p21 Waf1/CIP1. AKT also plays a critical role in the regulation of TORC1 and tuberin (TSC2).
Abcam has not and does not intend to apply for the REACH Authorisation of customers’ uses of products that contain European Authorisation list (Annex XIV) substances.
It is the responsibility of our customers to check the necessity of application of REACH Authorisation, and any other relevant authorisations, for their intended uses.
Store at +4°C. Please refer to protocols.
Components 1 x 96 tests 10X Wash Buffer PT 1 x 15ml 50X Cell Extraction Enhancer Solution 1 x 1ml 5X Cell Extraction Buffer PTR 1 x 12ml AKT 1/2/3 Total Capture Antibody 1 x 3ml AKT 1/2/3 Total Detector Antibody 1 x 3ml Lyophilized AKT Control Lysate 1 vial Plate Seal 1 unit SimpleStep Pre-Coated 96-Well Microplate (ab206978) 1 unit Stop Solution 1 x 12ml TMB Substrate 1 x 12ml
- Transcription factors
AKT2 is one of 3 closely related serine/threonine-protein kinases (AKT1, AKT2 and AKT3) called the AKT kinase, and which regulate many processes including metabolism, proliferation, cell survival, growth and angiogenesis. This is mediated through serine and/or threonine phosphorylation of a range of downstream substrates. Over 100 substrate candidates have been reported so far, but for most of them, no isoform specificity has been reported. AKT is responsible of the regulation of glucose uptake by mediating insulin-induced translocation of the SLC2A4/GLUT4 glucose transporter to the cell surface. Phosphorylation of PTPN1 at 'Ser-50' negatively modulates its phosphatase activity preventing dephosphorylation of the insulin receptor and the attenuation of insulin signaling. Phosphorylation of TBC1D4 triggers the binding of this effector to inhibitory 14-3-3 proteins, which is required for insulin-stimulated glucose transport. AKT regulates also the storage of glucose in the form of glycogen by phosphorylating GSK3A at 'Ser-21' and GSK3B at 'Ser-9', resulting in inhibition of its kinase activity. Phosphorylation of GSK3 isoforms by AKT is also thought to be one mechanism by which cell proliferation is driven. AKT regulates also cell survival via the phosphorylation of MAP3K5 (apoptosis signal-related kinase). Phosphorylation of 'Ser-83' decreases MAP3K5 kinase activity stimulated by oxidative stress and thereby prevents apoptosis. AKT 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. AKT is involved in the phosphorylation of members of the FOXO factors (Forkhead family of transcription factors), leading to binding of 14-3-3 proteins and cytoplasmic localization. In particular, FOXO1 is phosphorylated at 'Thr-24', 'Ser-256' and 'Ser-319'. FOXO3 and FOXO4 are phosphorylated on equivalent sites. AKT has an important role in the regulation of NF-kappa-B-dependent gene transcription and positively regulates the activity of CREB1 (cyclic AMP (cAMP)-response element binding protein). The phosphorylation of CREB1 induces the binding of accessory proteins that are necessary for the transcription of pro-survival genes such as BCL2 and MCL1. AKT phosphorylates 'Ser-454' on ATP citrate lyase (ACLY), thereby potentially regulating ACLY activity and fatty acid synthesis. Activates the 3B isoform of cyclic nucleotide phosphodiesterase (PDE3B) via phosphorylation of 'Ser-273', resulting in reduced cyclic AMP levels and inhibition of lipolysis. Phosphorylates PIKFYVE on 'Ser-318', which results in increased PI3P-5 activity. The Rho GTPase-activating protein DLC1 is another substrate and its phosphorylation is implicated in the regulation cell proliferation and cell growth. AKT plays a role as 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. Signals downstream of phosphatidylinositol 3-kinase (PI3K) 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). AKT mediates the antiapoptotic effects of IGF-I. Essential for the SPATA13-mediated regulation of cell migration and adhesion assembly and disassembly. May be involved in the regulation of the placental development.
Cytoplasm. Nucleus. Cell membrane.
- Entrez Gene: 10000 Human
- Entrez Gene: 207 Human
- Entrez Gene: 208 Human
- Entrez Gene: 11652 Mouse
- Entrez Gene: 11651 Mouse
- Entrez Gene: 23797 Mouse
- Entrez Gene: 25233 Rat
- Entrez Gene: 24185 Rat
Other - AKT 1/2/3 Total ELISA Kit (ab253298)
SimpleStep ELISA technology allows the formation of the antibody-antigen complex in one single step, reducing assay time to 90 minutes. Add samples or standards and antibody mix to wells all at once, incubate, wash, and add your final substrate. See protocol for a detailed step-by-step guide.
Example of a typical AKT 1/2/3 Total control lysate dilution series in 1X Cell Extraction Buffer PTR.
Raw data values are shown in the table.
Example of a typical AKT1 Total recombinant protein standard curve.
Background-subtracted data values (mean +/- SD) are graphed.
Cell line analysis for AKT 1/2/3 Total from preparations of 17,500 cells/well cell extracts.
Data from triplicate measurements (mean +/- SD) are plotted.
Induction of AKT 1/2/3 (pS473) phosphorylation in MCF-7 cells in response to insulin treatment, and levels of AKT 1/2/3 Total in the same samples.
MCF-7 cells were cultured in 96-well tissue culture plates, serum-starved (2 hr) and treated (30 min) with a dose-range of insulin before cell lysis. Data from duplicate measurements of AKT 1/2/3 (pS473) are plotted and compared against AKT 1/2/3 Total protein levels.
- Protocol Booklet
Click here to view the general protocols
Datasheets and documents
Publishing research using ab253298? Please let us know so that we can cite the reference in this datasheet.
ab253298 has not yet been referenced specifically in any publications.