Overview

  • Product name

    Human STAT5 (Tyr694) In-Cell ELISA Kit
    See all STAT5 kits
  • Detection method

    Colorimetric
  • Sample type

    Adherent cells
  • Assay type

    Semi-quantitative
  • Assay time

    5h 10m
  • Assay duration

    Multiple steps standard assay
  • Species reactivity

    Reacts with: Human
  • Product overview

    ab126429 is a very rapid, convenient and sensitive assay kit that can monitor the activation or function of important biological pathways in cells. It can be used for measuring the relative amount of STAT5 (Tyr694) phosphorylation and screening the effects of various treatments, inhibitors (such as siRNA or chemicals), or activators in cultured human cell lines. By determining STAT5 protein phosphorylation in your experimental model system, you can verify pathway activation in your cell lines without spending excess time and effort in preparing cell lysate and performing an analysis of Western Blot.


    In the In-Cell STAT5 (Tyr694) ELISA kit, cells are seeded into a 96 well tissue culture plate. The cells are fixed after various treatments, inhibitors or activators. After blocking, Anti Phospho-STAT5 (Tyr694) or Anti-STAT5 (primary antibody) is pipetted into the wells and incubated. The wells are washed, and HRP-conjugated anti-mouse IgG (secondary antibody) is added to the wells. The wells are washed again, a TMB substrate solution is added to the wells and color develops in proportion to the amount of protein. The Stop Solution changes the color from blue to yellow, and the intensity of the color is measured at 450 nm.

  • Tested applications

    Suitable for: In-Cell ELISAmore details
  • Platform

    Microplate

Properties

Applications

Our Abpromise guarantee covers the use of ab126429 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
In-Cell ELISA Use at an assay dependent concentration.

Images

  • A431 cells were stimulated by different concentrations of EGF for 10 minutes at 37°C.

     

  • A431 cells were stimulated by different concentrations of EGF for 30 minutes at 37°C.

  • Western blot analysis of extracts from 100 ng/ml hEGF treated A431 cells. Phospho-STAT5 (Tyr694) and STAT5 antibodies were used in both detection assays.
  • A431 cells were stimulated by different concentrations of EGF for 10 minutes at 37°C.
  • A431 cells were stimulated by different concentrations of EGF for 30 minutes at 37°C.

Protocols

References

ab126429 has not yet been referenced specifically in any publications.

Customer reviews and Q&As

Answer

We have not tried the assay with non-adherent cells and we are not aware of any other lab trying this either. It might be accomplished by centrifuging the cells in a 96-well plate after each wash, to pull them down into pellets in each well, but you would likely have some cell loss during aspiration of the washes, and possibly some loss due to cell lysis from the centrifugation. You could check to see how much by testing centrifugation of your cells in a plate (assuming you have plate holders) a few times, without actually doing the assay.

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