Overview

  • Product name
    STAT3 (Tyr705) In-Cell ELISA Kit
    See all STAT3 kits
  • Detection method
    Colorimetric
  • Sample type
    Adherent cells
  • Assay type
    Cell-based (qualitative)
  • Assay time
    5h 10m
  • Assay duration
    Multiple steps standard assay
  • Species reactivity
    Reacts with: Mouse, Rat, Human
  • Product overview

    ab126427 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 STAT3 (Tyr705) phosphorylation and screening the effects of various treatments, inhibitors (such as siRNA or chemicals), or activators in cultured human, mouse and rat cell lines. By determining STAT3 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 STAT3 (Tyr705) In-Cell 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-STAT3 (Tyr705) or Anti-STAT3 is pipetted into the wells and incubated. The wells are washed, and HRP-conjugated anti-mouse IgG 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

  • Storage instructions
    Store at -20°C. Please refer to protocols.
  • Components 1 x 96 tests
    HRP-conjugated Anti-Mouse IgG Concentrate 1 x 10µl
    Blocking Buffer Concentrate (5X) 1 x 20ml
    Fixing Solution 1 x 30ml
    Uncoated 96-well Microplate 1 unit
    Mouse Anti-Phospho-STAT3 (Tyr705) Concentrate (Item G) 1 x 10µl
    Mouse Anti-STAT3 Concentrate (Item H) 1 x 10µl
    Quenching Buffer Concentrate (30x) 1 x 2ml
    Stop Solution 1 x 14ml
    TMB One-Step Substrate Reagent 1 x 12ml
    Wash Buffer A Concentrate (20X) 1 x 30ml
    Wash Buffer B Concentrate (20X) 1 x 30ml
  • Research areas
  • Function
    Signal transducer and transcription activator that mediates cellular responses to interleukins, KITLG/SCF, LEP and other growth factors. Once activated, recruits coactivators, such as NCOA1 or MED1, to the promoter region of the target gene (PubMed:17344214). May mediate cellular responses to activated FGFR1, FGFR2, FGFR3 and FGFR4. Binds to the interleukin-6 (IL-6)-responsive elements identified in the promoters of various acute-phase protein genes. Activated by IL31 through IL31RA. Involved in cell cycle regulation by inducing the expression of key genes for the progression from G1 to S phase, such as CCND1 (PubMed:17344214). Mediates the effects of LEP on melanocortin production, body energy homeostasis and lactation (By similarity). May play an apoptotic role by transctivating BIRC5 expression under LEP activation (PubMed:18242580). Cytoplasmic STAT3 represses macroautophagy by inhibiting EIF2AK2/PKR activity.
  • Tissue specificity
    Heart, brain, placenta, lung, liver, skeletal muscle, kidney and pancreas.
  • Involvement in disease
    Hyperimmunoglobulin E recurrent infection syndrome, autosomal dominant
    Autoimmune disease, multisystem, infantile-onset
  • Sequence similarities
    Belongs to the transcription factor STAT family.
    Contains 1 SH2 domain.
  • Post-translational
    modifications
    Tyrosine phosphorylated upon stimulation with EGF. Tyrosine phosphorylated in response to constitutively activated FGFR1, FGFR2, FGFR3 and FGFR4 (By similarity). Activated through tyrosine phosphorylation by BMX. Tyrosine phosphorylated in response to IL6, IL11, LIF, CNTF, KITLG/SCF, CSF1, EGF, PDGF, IFN-alpha, LEP and OSM. Activated KIT promotes phosphorylation on tyrosine residues and subsequent translocation to the nucleus. Phosphorylated on serine upon DNA damage, probably by ATM or ATR. Serine phosphorylation is important for the formation of stable DNA-binding STAT3 homodimers and maximal transcriptional activity. ARL2BP may participate in keeping the phosphorylated state of STAT3 within the nucleus. Upon LPS challenge, phosphorylated within the nucleus by IRAK1. Upon erythropoietin treatment, phosphorylated on Ser-727 by RPS6KA5. Phosphorylation at Tyr-705 by PTK6 or FER leads to an increase of its transcriptional activity. Dephosphorylation on tyrosine residues by PTPN2 negatively regulates IL6/interleukin-6 signaling.
  • Cellular localization
    Cytoplasm. Nucleus. Shuttles between the nucleus and the cytoplasm. Translocated into the nucleus upon tyrosine phosphorylation and dimerization, in response to signaling by activated FGFR1, FGFR2, FGFR3 or FGFR4. Constitutive nuclear presence is independent of tyrosine phosphorylation. Predominantly present in the cytoplasm without stimuli. Upon leukemia inhibitory factor (LIF) stimulation, accumulates in the nucleus. The complex composed of BART and ARL2 plays an important role in the nuclear translocation and retention of STAT3. Identified in a complex with LYN and PAG1.
  • Information by UniProt
  • Alternative names
    • 1110034C02Rik
    • Acute Phase Response Factor
    • Acute-phase response factor
    • ADMIO
    • APRF
    • AW109958
    • DNA binding protein APRF
    • FLJ20882
    • HIES
    • MGC16063
    • Signal transducer and activator of transcription 3
    • Signal transducer and activator of transcription 3 (acute phase response factor)
    • STAT 3
    • Stat3
    • STAT3_HUMAN
    see all
  • Database links

Applications

Our Abpromise guarantee covers the use of ab126427 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 30 minutes at 37°C.

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

  • Western blot analysis of extracts from 100 ng/ml hEGF treated A431 cells. Phospho-Stat3 (Tyr705) and Anti-Stat3 antibodies were used in both detection assays.
  • A431 cells were stimulated by different concentrations of EGF for 30 minutes at 37°C.

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

Protocols

References

ab126427 has not yet been referenced specifically in any publications.

Customer reviews and Q&As

Answer

Thank you for your recent telephone enquiry and for your patience waiting for a response. It has taken some time to confirm these details with the originator of this kit.

I can confirm the plate provided with this kit are not precoated, and you will need to pre-coat the 96 well microplate (Item A) by adding 100 µl poly-L-Lysine into each well. We highly recommend using the Microplate provided for this assay.

The assay should work fine for rat primary neuronal cells, providing that the cells are prepared correctly. The assay protocol is provided on the onlinedatasheet:

https://www.abcam.com/ps/products/126/ab126427/documents/STAT3%20_Tyr705_%20In-Cell%20ELISA%20Kit-ab126427-protocol-v2%20(Web).pdf

I am also pleased to provide the followinginformation regarding sample preparation:

The cell or tissue lysates forfor use with this kit can be prepared using most conventional methods, e.g. homogenization of cell or tissue in Lysis Buffer. We supply a compatible lysis buffer in most of our kits. Other general low-salt lysis buffers can be used with the following caveats:

1) Avoid using SDS or other strongly denaturing detergents. In general, non-ionic detergents, such as Triton X-100 or NP-40 are best, although zwitterionic detergents, such as CHAPS, or mild ionic detergents, such as sodium deoxycholate will work.
2) Use no more than 2% v/v total detergent
3) Avoid the use of sodium azide
4) Avoid reducing agents, such as dithiothreitol or mercaptoethanols

In general, we strongly recommend that some type of protease inhibitor cocktail is included in the lysis buffer prior to homogenization.

Since susceptibility to proteolytic cleavage and the type of proteases present in the lysate vary, we do not recommend a specific product. Instead, your choice of which combination of protease inhibitors to use should be based upon a literature search for your protein(s) of interest and/or tissue or cell type. Phosphatase inhibitors may be used, but are not necessary unless the antibodies used in the kit specifically recognize phosphorylated (activated) forms of the protein.

Choices of the method for lysis and homogenization include glass-bead, douncing, freeze-thaw, sonication and crushing frozen tissue with a mortar and pestle, or even a combination of these. There is no one best method; for all sample types, but some are better than others for some sample types. Your choice of method should be made following a brief search of the literature to see how samples similar to yours have been prepared in previous investigations.

After homogenization, spin down the lysates to remove cell/tissue debris (5 min @ 10000 x g or 10 min @ 5000 x g) and save the supernatant. Lysates should be frozen as soon as possible, and stored at -20°C (or -80°C, if possible). Spin them again before incubating with the antibody array. Determine the protein concentration of your lysates (for example the bicinchoninic acid (BCA) assay) and normalize the volume of each sample used to deliver the same amount of total protein for each assay.

Since different cells and tissues may contain different amounts of protein, as starting point, we suggest using 500 uL of lysis buffer per 1x10e6 cells or 10 mg tissue. You may have to adjust this based upon your results. Your target for total protein concentration of the homogenate should be at least 1000 ug/mL, but 2000 ug/mL would be better.

There is also a reference in which the kit has been used to test rat neuronal cells (shown below).Regrettably, I'm not able togain full access to the article without an account with the journal database. But I hope this will be helpful:

http://www.sciencedirect.com/science/article/pii/S0006899310026090

If you have any further questions, please do not hesitate to contactme.

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