Overview

  • Product name

    Phospho-STAT6 (Y641) + Total In-Cell ELISA Kit (Chemiluminescent)
    See all STAT6 kits
  • Detection method

    Luminescent
  • Sample type

    Adherent cells, Suspension cells
  • Assay type

    Cell-based (quantitative)
  • Assay duration

    Multiple steps standard assay
  • Species reactivity

    Reacts with: Mouse, Human
  • Product overview

    Phospho-STAT6 (Y641) + Total In-Cell ELISA Kit (Chemiluminescent) (ab207489) provides a simple, efficient, cell-based method to monitor proteins activated by phosphorylation. The kit is designed specifically to quantify activated (phosphorylated) STAT6 and/or total STAT6. Cells are cultured in 96-well plates and stimulated to induce the pathway of interest. Following stimulation, the cells are rapidly fixed to preserve activation-specific protein modifications. Each well is then incubated with a primary antibody that recognizes either phosphorylated STAT6 or total STAT6. Subsequent incubation with secondary HRP-conjugated antibody and developing solution provides an easily quantified chemiluminescent readout. The relative number of cells in each well is then determined using the provided Crystal Violet solution. The 96-well plate format is suitable for high-throughput screening applications.


    The Phospho-STAT6 (Y641) + Total In-Cell ELISA Kit (Chemiluminescent) contains two 96-well plates and two primary antibodies. The phospho-STAT6 antibody is specific for phosphorylated STAT6 and was raised against a synthetic phospho-peptide corresponding to residues surrounding Tyrosine 641 of mouse STAT6. The total-STAT6 antibody recognizes STAT6 proteins regardless of the phosphorylation state. The kit can be used to study phosphorylated STAT6 relative to cell number or to determine STAT6 phosphorylation relative to the total STAT6 protein found in the cells. Once the phospho-STAT6 and total-STAT6 signals have been normalized for cell number, a comparison of the ratio of phosphorylated STAT6 to total STAT6 for each of the cell growth conditions can be made. The provided total-STAT6 antibody can be used as a positive control to demonstrate that the cells contain STAT6, the kit reagents are functional and that the protocol is performed correctly. Also, because fixed cells are stable for several weeks, you can prepare many plates simultaneously and then perform the assay when desired.

  • Notes

    STAT (signal transducers and activators of transcription) comprise a family of latent cytoplasmic proteins that are activated to participate in gene control when cells encounter various extracellular polypeptides. Their critical role in development and normal cell signaling has been largely determined through the analysis of transgenic mice lacking individual STAT genes.

    The STAT proteins are unique among transcription factors in containing an SH2 (src-homology 2), phosphotyrosine-binding domain, a common protein-protein interaction domain among signaling proteins. Tyrosine phosphorylation around residue 700 is essential for the dimerization of STATs and the concomitant nuclear translocation of the dimer. Ligand-activated receptors that catalyze this phosphorylation include receptors with intrinsic tyrosine kinase activity (epidermal growth factor (EGF), platelet-derived growth factor (PDGF) and colony-stimulating factor-1) as well as receptors that lack intrinsic tyrosine kinase activity but to which Janus kinases (JAKs) are noncovalently associated. Receptors to which JAKs are bound are often referred to as cytokine receptors. Their ligands include IFN-α, -β and -γ; interleukins (IL) 2 to 7, 10 to 13, and 15; and erythropoietin, growth hormone, prolactin, thrombopoietin and other polypeptides. STAT dimers and heterodimers, but not monomers, are competent to bind DNA. The known DNA binding heterodimers are STAT1:2 (strong binding requires the joint presence of another protein, p48) and STAT1:3. STATs that form homodimers that bind DNA include STAT 1, 3, 4, 5 (STAT5A and 5B interact in a manner equivalent to a heterodimer) and 6.

    STAT proteins are involved in a wide variety of biological pathways. STAT1 is involved in the activation of IFNα and IFNγ genes, STAT2 in the activation of IFNα genes, STAT4 and STAT6 in T-helper cell development and STAT5 in milk production. Disruption of STAT functions in mouse leads to several defects such as immune deficiency (STAT1), embryonic lethality (STAT2), lack of gastrulation (STAT3), T-helper 1 cell dysfunction (STAT4), lack of lactation (STAT5A, 5B) and T-helper 2 cell dysfunction (STAT6). The disruption of STAT signaling blocks neoplastic transformation, thus making inhibitors of STAT proteins candidates for the treatment of cancer.

    In most cases, STAT activation is transient. Inactivation of STAT proteins is carried out by several mechanisms, including dephosphorylation of STAT proteins in the nucleus and degradation through the ubiquitin-proteosome pathway. A novel family of negative feedback inhibitors of the JAK-STAT pathway has been identified, referred to as suppressor-of-cytokine-signaling (SOCS) proteins/JAK binding (JAB) proteins, and STAT-induced STAT inhibitors (SSIs).

  • Tested applications

    Suitable for: In-Cell ELISAmore details
  • Platform

    Microplate

Properties

Applications

Our Abpromise guarantee covers the use of ab207489 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

  • NIH/3T3 cells were cultured in 96-well plates and serum-starved for 16 hours. Cells were then treated with 50 ng/mL PDGF for 5 minutes and fixed. Total and phospho STAT6, was each assayed in triplicate using the phospho and total STAT6 antibodies included in the kit. Data was plotted after correction for cell number (performed through use of Crystal Violet)

Protocols

References

ab207489 has not yet been referenced specifically in any publications.

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