• Product name
  • Detection method
  • Precision
    Sample n Mean SD CV%
    AKT (pS473) 6 3.2%
    AKT1 (Total) 6 4%
    Sample n Mean SD CV%
    AKT (pS473) 3 2.7%
    AKT1 (Total) 3 4.9%
  • Sample type
    Cell Lysate, Tissue Homogenate
  • Assay type
  • Sensitivity
    30 pg/ml
  • Assay time
    1h 30m
  • Assay duration
    One step assay
  • Species reactivity
    Reacts with: Mouse, Rat, Human
  • Product overview

    Abcam’s AKT 1/2/3 (pS473) and AKT1 (Total) in vitro SimpleStep™ ELISA (Enzyme-Linked Immunosorbent Assay) kit is designed for the semi-quantitative measurement of AKT 1/2/3 (pS473) and total AKT1 protein in Human and mouse cells.

    The SimpleStep ELISA™ employs an affinity tag labeled capture antibody and a reporter conjugated detector antibody which immunocapture the sample analyte in solution. This entire complex (capture antibody/analyte/detector antibody) is in turn immobilized via immunoaffinity of an anti-tag antibody coating the well. To perform the assay, samples or standards are added to the wells, followed by the antibody mix. After incubation, the wells are washed to remove unbound material. TMB substrate is added and during incubation is catalyzed by HRP, generating blue coloration. This reaction is then stopped by addition of Stop Solution completing any color change from blue to yellow. Signal is generated proportionally to the amount of bound analyte and the intensity is measured at 450 nm. Optionally, instead of the endpoint reading, development of TMB can be recorded kinetically at 600 nm.

  • Notes

    Estimated sensitivity: Phospho-AKT1/2/3 (Ser473): 0.5 ng/mL (tested with recombinant protein), Total AKT1: 0.2 ng/mL (tested with recombinant protein)
    Range: Phospho-AKT1/2/3 (Ser473): 1-100 ng/mL, Total AKT1: 0.5-50 ng/mL

  • Tested applications
    Suitable for: Sandwich ELISAmore details
  • Platform


Associated products


Our Abpromise guarantee covers the use of ab176657 in the following tested applications.

The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.

Application Abreviews Notes
Sandwich ELISA Use at an assay dependent concentration.


  • Example of a typical AKT (pS473) and AKT1 (Total) cell lysate dilution series. Background-subtracted data values (mean +/- SD) are graphed.

  • Linearity of dilution in representative sample matrices. Cellular lysates were prepared at 3 concentrations in common media containing 1X Cell Extraction Buffer PTR. Data from duplicate measurements of AKT1 (pS473) are normalized and plotted.

  • Cell line analysis for total AKT1 from 20 µg/mL preparations of cell extracts. Data from triplicate measurements (mean +/- SD) are plotted and compared to 1X Cell Extraction Buffer PTR (zero).

  • Induction of AKT (pS473) phosphorylation in MCF-7 cells in response to insulin treatment. MCF-7 cells were cultured in 96-well tissue culture plates, serum-starved and treated (5 min) with a dose-range of insulin before cell lysis. Data from quadruplicate measurements of AKT (pS473) are plotted and compared against total AKT1 protein levels. Comparative AKT (pS473) and AKT1 (Total) data also shown by Western Blot.



This product has been referenced in:
  • Wierzbicki M  et al. Diamond, graphite, and graphene oxide nanoparticles decrease migration and invasiveness in glioblastoma cell lines by impairing extracellular adhesion. Int J Nanomedicine 12:7241-7254 (2017). Read more (PubMed: 29042773) »
See 1 Publication for this product

Customer reviews and Q&As

Akt SimpleStep kit

Good Good 4/5 (Ease of Use)
The AKT 1/2/3 (pS473) + AKT1 (Total) SimpleStep ELISA™ Kit (ab176657) was user-friendly, and straight forward in the method and user manual. The only suggestion I would make is to provide clearer direction to the user that the kit only does 1 sample, twice. I.e., half of the wells are for the total Akt, and the other half are for p-Akt. This will allow the user to order multiple kits at one time, since the kit does not hold as many samples as a typical ELISA kit does (96 samples). This kit in fact only allows for 48 samples, run twice. Tested in rat muscle lysate.

Kaitlin Giles

Verified customer

Submitted Aug 20 2014


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