-->

Total STAT3 cellular kit HTRF®

This HTRF kit detects cellular STAT3 and can be used as a normalization assay with our phospho-STAT3 kit for an optimal readout of JAK/STAT signaling.

See more
  • Ready-to-use Ready-to-use
  • High sensitivity High sensitivity
  • Faster and more convenient than ELISA Faster and more convenient than ELISA
  • Low sample consumption Low sample consumption

This HTRF kit detects cellular STAT3 and can be used as a normalization assay with our phospho-STAT3 kit for an optimal readout of JAK/STAT signaling.

-
In stock

Overview

The Total STAT3 cellular assay kit is designed to monitor the expression level of STAT3, whether phosphorylated or unphosphorylated. It is compatible with our Phospho-STAT3 kit, and enables the analysis of phosphorylated and total proteins from a single sample for better readout of the JAK/STAT signaling pathway.

Benefits

  • SPECIFICITY
  • PRECISION
  • DATA NORMALIZATION

Total-STAT3 assay principle

The Total-STAT3 assay quantifies the expression level of STAT3 in a cell lysate. Contrary to Western Blot, the assay is entirely plate-based and does not require gels, electrophoresis or transfer. The Total-STAT3 assay uses two labeled antibodies: one coupled to a donor fluorophore, the other to an acceptor. Both antibodies are highly specific for a distinct epitope on the protein. In presence of STAT3 in a cell extract, the addition of these conjugates brings the donor fluorophore into close proximity with the acceptor and thereby generates a FRET signal. Its intensity is directly proportional to the concentration of the protein present in the sample, and provides a means of assessing the protein’s expression under a no-wash assay format.
Total-STAT3 assay principle

Total-STAT3 2-plate assay protocol

The 2 plate protocol involves culturing cells in a 96-well plate before lysis then transferring lysates to a 384-well low volume detection plate before adding Total-STAT3 HTRF detection reagents. This protocol enables the cells' viability and confluence to be monitored.
Total-STAT3 2-plate assay protocol

Total-STAT3 1-plate assay protocol

Detection of total STAT3 with HTRF reagents can be performed in a single plate used for culturing, stimulation and lysis. No washing steps are required. This HTS designed protocol enables miniaturization while maintaining robust HTRF quality.
Total-STAT3 1-plate assay protocol

HTRF compared to Western Blot using total-STAT3 assay

HEK293 cells were grown in a T175 flask at 37°C, 5% CO2 for 2 days. After removal of cell culture medium, 3mL of supplemented lysis buffer were added and incubated for 45 min. Soluble supernatants were collected after 10 min centrifuging. Equal amounts of lysates were used for a side by side comparison of WB and HTRF. 150,000 cells correspond to approximately 20µg of total protein. The two methods show a comparable sensitivity level: 1172 cells could be detected with each technology.
HTRF compared to Western Blot using total-STAT3 assay

Validation of the total-STAT3 kit on various species of cell lines: human, mouse and hamster

Human (Hela, A431, HEK293, Jurkat), murine (NIH 3T3) and Hamster (CHO-K1) cells were grown in a T175 flask at 37°C, 5% CO2 for 2 days. After removal of cell culture medium, 3mL of supplemented lysis buffer 3 were added and incubated for 45 min. Soluble supernatants were collected after 10 min centrifuging. STAT3 was detected using 16µL of cell lysate.

Validation of the total-STAT3 kit on various species of cell lines: human, mouse and hamster

HTRF total-STAT3 assay used to check the phosphorylation status of STAT3

Hela cells (100,000 cells/well) were activated with IFNa for 15 min, using the two-plate assay protocol of the Phospho-STAT3 (Tyr705) and Total-STAT3 assays. Results obtained show a dose-response increase of STAT3 phosphorylation upon IFNα stimulation, while STAT3 expression level remains constant.

HTRF total-STAT3 assay used to check the phosphorylation status of STAT3

STAT3 Epidermal growth factor signaling

In response to cytokines and growth factors, STAT3 is phosphorylated by receptor-associated kinases and then forms dimers that translocate to the cell nucleus, where they act as transcription activators. STAT3 mediates the expression of a variety of genes in response to cell stimuli, and thus plays a key role in many cellular processes such as cell growth and apoptosis. The binding of Interleukin 6-family cytokines to the gp130 receptor triggers STAT3 phosphorylation by JAK2. STAT3 is also a target of other receptors such as RTKs (EGFR, cmet.) and c-src.

STAT3 Epidermal growth factor signaling pathway

Simplified pathway dissection with HTRF phospho-assays and CyBi-felix liquid handling

Analyse of PI3K/AKT/mTor translational control pathway - Application Notes

Cisbio lysis buffer compatibility

Cell Signaling: Biomarkers, Phospho- & total-protein Assays - Flyers

HTRF cellular phospho-protein assays

Physiologically relevant results fo fast flowing research - Flyers

Inflammation cell by cell

HTRF solutions for each cell type - Flyers

Species compatibility

Cell Signaling: Biomarkers, Phospho- & total-protein assays - Flyers

Universal HTRF® phospho-protein platform: from 2D, 3D, primary cells to patient derived tumor cells

Analysis of a large panel of diverse biological samples and cellular models - Posters

HTRF phospho assays reveal subtle drug induced effects in tumor-xenografts

Tumor xenograft analysis: HTRF versus Western blot - Application Notes

HTRF cell-based phospho-protein data normalization

Valuable guidelines for efficiently analyzing and interpreting results - Application Notes

HTRF phospho-total lysis buffer: a universal alternative to RIPA lysis buffers

Increased flexibility of phospho-assays - Application Notes

Best practices for analyzing brain samples with HTRF® phospho assays for neurosciences

Insider Tips for successful sample treatment - Technical Notes

HTRF Alpha-tubulin Housekeeping kit

Properly interpret your compound effect - Application Notes

Optimize your HTRF cell signaling assays on tissues

HTRF and WB compatible guidelines - Technical Notes

Key guidelines to successful cell signaling experiments

Mastering the art of cell signaling assays optimization - Guides

HTRF phospho-assays reveal subtle drug-induced effects

Detailed protocol and direct comparison with WB - Posters

Best practices for analyzing tumor xenografts with HTRF phospho assays

Protocol for tumor xenograft analysis with HTRF - Technical Notes

How to run a cell based phospho HTRF assay

What to expect at the bench - Videos

Unleash the potential of your phosphorylation research with HTRF

Unmatched ease of use, sensitivity and specificity assays - Videos

Product Insert STAT3 total Kit / 64NT3PEG-64NT3PEH

64NT3PEG-64NT3PEH - Product Insert

HTRF Product Catalog 2020 July update

All your HTRF assays in one document! - Catalog

A guide to Homogeneous Time Resolved Fluorescence

General principles of HTRF - Guides

How HTRF compares to Western Blot and ELISA

Get the brochure about technology comparison. - Brochures

HTRF® cell signaling platform combined with iCell® Hepatocytes

A solution for phospho-protein analysis in metabolic disorders - Posters

Unleash the potential of your phosphorylation research with HTRF

A fun video introducing you to phosphorylation assays with HTRF - Videos

How to run a cell based phospho HTRF assay

3' video to set up your Phospho assay - Videos

Plate Reader Requirement

Choosing the right microplate reader ensures you’ll get an optimal readout. Discover our high performance reader, or verify if your lab equipment is going to be compatible with this detection technology.

Let's find your reader

Latest news