Phospho-STAT3 (Tyr705) cellular kit
All-in-one kit for robust detection of Phospho-STAT3 (Ser423/425)
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.
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Ready-to-use
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High sensitivity
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Faster and more convenient than ELISA
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Low sample consumption
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 proteins expression under a no-wash assay format.
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 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.
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.
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.
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.
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.
Simplified pathway dissection with HTRF phospho-assays and CyBi-felix liquid handling
Analyse of PI3K/AKT/mTor translational control pathway - Application Notes
Cell Signaling: Biomarkers, Phospho- & total-protein Assays - Flyers
HTRF cellular phospho-protein assays
Physiologically relevant results fo fast flowing research - Flyers
Biomarker and Cell Signaling Assays for Fibrosis and NASH
HTRF and Alpha solutions for NASH - Flyers
HTRF solutions for each cell type - Flyers
Cell Signaling: Biomarkers, Phospho- & total-protein assays - Flyers
PI3K/AKT/mTor translational control pathway - Posters
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
From 2D, 3D cell cultures to xenografts: A smart HTRF platform to maximize anticancer drug discovery
One technology across all samples - Application Notes
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
Multi-tissue cellular modeling and anlysis of insulin signaling - Posters
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
A single technology for 2D cells, 3D cells, and xenograft models - 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
Cell-based kinase assays in HTS ? potential and limitations for primary and secondary screening
In collaboration with Bayer - Scientific Presentations
Product Insert STAT3 total Kit / 64NT3PEG-64NT3PEH
64NT3PEG-64NT3PEH - Product Insert
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
Advance your research on Fibrosis
Kits and reagents for Fibrosis research - Flyers
Guidelines for Cell Culture and Lysis in Different Formats Prior to HTRF Detection
Seeding and lysing recommendations for a number of cell culture vessels. - Technical Notes
Assessment of drug efficacy and toxicity by combining innovative technologies
Combination of AlphaLISA®, HTRF®, or AlphaLISA® SureFire® Ultra™ immunoassays with the ATPlite™ 1step cell viability assay - Application Notes
Methodological Aspects of Homogeneous Time-Resolved Fluorescence (HTRF)
Learn how to reduce time and sample consumption - Application Notes
Safety Data Sheet (DEU) STAT3 total Kit / 64NT3PEG
64NT3PEG - Safety Data Sheet
Safety Data Sheet (ELL) STAT3 total Kit / 64NT3PEG
64NT3PEG - Safety Data Sheet
Safety Data Sheet (FRA-FR) STAT3 total Kit / 64NT3PEG
64NT3PEG - Safety Data Sheet
Safety Data Sheet (ITA) STAT3 total Kit / 64NT3PEG
64NT3PEG - Safety Data Sheet
Safety Data Sheet (SPA) STAT3 total Kit / 64NT3PEG
64NT3PEG - Safety Data Sheet
Safety Data Sheet (ENG-GB) STAT3 total Kit / 64NT3PEG
64NT3PEG - Safety Data Sheet
Safety Data Sheet (ENG-US) STAT3 total Kit / 64NT3PEG
64NT3PEG - Safety Data Sheet
Safety Data Sheet (DEU) STAT3 total Kit / 64NT3PEH
64NT3PEH - Safety Data Sheet
Safety Data Sheet (ELL) STAT3 total Kit / 64NT3PEH
64NT3PEH - Safety Data Sheet
Safety Data Sheet (FRA-FR) STAT3 total Kit / 64NT3PEH
64NT3PEH - Safety Data Sheet
Safety Data Sheet (ITA) STAT3 total Kit / 64NT3PEH
64NT3PEH - Safety Data Sheet
Safety Data Sheet (SPA) STAT3 total Kit / 64NT3PEH
64NT3PEH - Safety Data Sheet
Safety Data Sheet (ENG-GB) STAT3 total Kit / 64NT3PEH
64NT3PEH - Safety Data Sheet
Safety Data Sheet (ENG-US) STAT3 total Kit / 64NT3PEH
64NT3PEH - Safety Data Sheet
Plate Reader Requirement
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