Overview

  • Product name
    AKT 1/2/3 (pS473) ELISA Kit
    See all AKT 1/2/3 (phospho-Ser473) kits
  • Detection method
    Colorimetric
  • Precision
    Intra-assay
    Sample n Mean SD CV%
    HEK lysate 6 = 5.4%
    Inter-assay
    Sample n Mean SD CV%
    HEK lysate 3 = 2.7%
  • Sample type
    Cell Lysate, Tissue Homogenate
  • Assay type
    Semi-quantitative
  • Sensitivity
    30 pg/ml
  • Range
    1 ng/ml - 100 ng/ml
  • Recovery

    4.2 %

  • Assay time
    1h 30m
  • Assay duration
    One step assay
  • Species reactivity
    Reacts with: Mouse, Human
    Predicted to work with: Rat
  • Product overview

    Abcam’s AKT 1/2/3 (pS473) in vitro SimpleStep ELISA™ (Enzyme-Linked Immunosorbent Assay) kit is designed for the semi-quantitative measurement of AKT 1/2/3 (pS473) 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.

  • Tested applications
    Suitable for: Sandwich ELISAmore details
  • Platform
    Microplate

Properties

Associated products

Applications

Our Abpromise guarantee covers the use of ab176635 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.

Images

  • Example of a typical AKT (pS473) 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.

  • 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.

  • 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).

Protocols

References

ab176635 has not yet been referenced specifically in any publications.

Customer reviews and Q&As

Answer



Similarly to the AKT protocol, for the p53 kit, 96WP samples can be processed. If this is the case, 0.1mL is an appropriate starting volume for lysing cells in a microplate. Extracts should be pipetted up/down several times to fully solubilize.

The main caution to take is to make certain that you can achieve a workable concentration of extract from the 96WP. Please have a look at Figures 2 &3 in the p53 Simple Step ELISA booklet (ab171571, page 20) as guidance for concentrations to be within the working range of the assay for some example cell lines.

I would recommend running a pilot experiment to determine what concentrations can be achieved for your given cell line and culture conditions – the results should then dictate the lysis volume and if the extract needs to be further diluted in extraction buffer before adding to the ELISA plate.

We think scraping increases the recovery of extract on large plates but it is not strictly necessary (and of course is impossible in 96WP).

It is also advisable to take care in washing cells in PBS if the treatment or culture conditions have caused any cells to become loosely attached. You may also want to consider normalizing extract loads to the ELISA plate (e.g. by BCA assay) if the treatments are causing an effect on cell number. Note that 50uL of sample always needs to be added to the SSE plate.

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Please note: All products are "FOR RESEARCH USE ONLY AND ARE NOT INTENDED FOR DIAGNOSTIC OR THERAPEUTIC USE"

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