Key features and details
- One-wash 90 minute protocol
- Sensitivity: 1 µg/ml
- Range: 0.05 % - 3.12 %
- Sample type: Cell Lysate
- Assay type: Semi-quantitative
- Reacts with: Mouse, Human
Product nameAKT 1/2/3 pS473 + AKT 1/2/3 Total ELISA Kit
Intra-assay Sample n Mean SD CV% AKT (pS473) 6 8.7% AKT Total 6 1.8% Inter-assay Sample n Mean SD CV% AKT (pS473) 3 5.3% AKT Total 3 9.5%
Sample typeCell Lysate
Sensitivity< 1 µg/ml
Range0.05 % - 3.12 %
Assay durationOne step assay
Species reactivityReacts with: Mouse, Human
Predicted to work with: Rat
AKT 1/2/3 pS473 + AKT 1/2/3 Total (ab253299) is a single-wash 90 min sandwich ELISA designed for the semi-quantitative measurement of total AKT 1/2/3 pS473 + AKT 1/2/3 total protein in human and mouse cell lysates. It uses our proprietary SimpleStep ELISA® technology. Semi-quantitate AKT 1/2/3 pS473 + AKT 1/2/3 total with 1 µg/mL sensitivity.
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.
PlatformPre-coated microplate (12 x 8 well strips)
Storage instructionsStore at +4°C. Please refer to protocols.
Components 1 x 96 tests 10X Wash Buffer PT 1 x 25ml 50X Cell Extraction Enhancer Solution 1 x 1ml 5X Cell Extraction Buffer PTR 1 x 12ml AKT 1/2/3 (pS473) Capture Antibody 1 x 1.5ml AKT 1/2/3 (pS473) Detector Antibody 1 x 1.5ml AKT 1/2/3 Total Capture Antibody 1 x 1.5ml AKT 1/2/3 Total Detector Antibody 1 x 1.5ml 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
- Epigenetics and Nuclear Signaling
- Domain Families
- HLH / Leucine Zipper
- HLH / Leucine Zipper
Cellular localizationAKT 1/2/3 (Total): Cytoplasm. Nucleus. Cell membrane.
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 AKT 1/2/3 (pS473) and AKT 1/2/3 Total control lysate dilution series in 1X Cell Extraction Buffer PTR.
Example of a typical AKT 1/2/3 (PS473) and AKT1 Total recombinant protein standard curve. The proportion of total protein that is phosphorylated is unknown - data is indicative only. Raw data values are shown in the table. Background-subtracted data values (mean +/- SD) are graphed.
Data from triplicate measurements (mean +/- SD) are plotted.
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.
ab253299 has not yet been referenced specifically in any publications.