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Phospho-STAT1 (Tyr701) cellular kit

Phospho-STAT1 (Tyr701) cellular kit HTRF®

This HTRF assay enables the cell-based quantitative detection of phosphorylated STAT1 on Tyr701 as a readout of the JAK/STAT pathway.

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

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This HTRF assay enables the cell-based quantitative detection of phosphorylated STAT1 on Tyr701 as a readout of the JAK/STAT pathway.

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Overview

This HTRF cell-based assay enables the rapid and quantitative detection of STAT1 phosphorylated on Tyr701 as a readout of JAK/STAT signaling activity. STAT1 acts as an important transcriptional activator and the assay serves as a readout for JAK inhibitors in oncology, virology and inflammation.

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Phospho-STAT1 (Tyr701) assay principle

The Phospho-STAT1 (Tyr701) assay measures STAT1 when phosphorylated at Tyr701. Contrary to Western Blot, the assay is entirely plate-based and does not require gels, electrophoresis or transfer. The Phospho-STAT1 (Tyr701) assay uses 2 labeled antibodies: one with a donor fluorophore, the other one with an acceptor. The first antibody is selected for its specific binding to the phosphorylated motif on the protein, the second for its ability to recognize the protein independent of its phosphorylation state. Protein phosphorylation enables an immune-complex formation involving both labeled antibodies and which brings the donor fluorophore into close proximity to the acceptor, thereby generating a FRET signal. Its intensity is directly proportional to the concentration of phosphorylated protein present in the sample, and provides a means of assessing the proteins phosphorylation state under a no-wash assay format.

Phospho-STAT1 (Tyr701) 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 phospho-STAT1 (Tyr701) HTRF detection reagents. This protocol enables the cells' viability and confluence to be monitored.

Phospho-STAT1 (Tyr701) Cellular Assay 2-plate assay protocol

Phospho-STAT1 (Tyr701) 1-plate assay protocol

Detection of Phosphorylated STAT1 (Tyr701) 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.

Phospho-STAT1 (Tyr701) Cellular Assay 1-plate protocol

HTRF assay compared to Western Blot using phospho-STAT1 cellular assays on human HeLa cells

HeLa cells were grown in a T175 flask at 37 °C, 5% CO2 for 48h. Cells were then stimulated with IFNa for 20 min. After medium removal, the cells were lysed with 3mL of supplemented lysis buffer for 30 min at room temperature. Soluble fractions were then collected after 10 min centrifugation. Serial dilutions of the cell lysate were performed in the supplemented lysis buffer and 16 µL of each dilution were dispensed and analyzed side-by-side by Western-blot and by HTRF. For each cell density tested, the fluorescence ratio closely matched the western blot band intensity, demonstrating the reliability of HTRF as an analytical technique for the measurement of phopsho STAT1

Validation of phospho STAT1-Y701 on human & murine cell lines

IFNa was used to induce phosphorylation of STAT1 in mouse NIH 3T3 and human HeLa cells. NIH-3T3 & Hela cells plated at different cell densities, were stimulated with 2.5µg/mL of IFNa for 20 minutes. After a lysis step using 50 µL of supplemented lysis buffer, 16 µL of lysate were transferred into a 384-well low volume white microplate before the addition of 4 µL of the HTRF phospho-STAT1 detection reagents. The HTRF signal was recorded after an overnight incubation. The graph display the biological assay window corresponding to IFNa-stimulated over unstimulated conditions.

Detection of phospho STAT1 on HeLa cells: stimulation kinetic

IFNa was used to induce phosphorylation of STAT1 in HeLa cells. 200,000 cells were plated in a 96well plate, then stimulated with increasing IFNa concentrations for various times (10, 20 30 and 60 minutes). After cell lysis with 50 µL of supplemented lysis buffer, 16 µL of lysate were transferred into a 384-well low volume white microplate before the addition of 4 µL of the HTRF phospho-STAT1 detection reagents. The HTRF signal was recorded after an overnight incubation. In these experimental conditions the optimal stimulation time was determined at 20 minutes of IFNa.

HTRF total-STAT1 assay used to check the phosphorylation status of STAT1 on murine and human cells

After 20 minutes stimulation with increasing IFNa concentrations, NIH 3T3 & Hela cells (200,000 cells/96well plate) were lysed with 50 µL of supplemented lysis buffer and incubated for 30 min at RT under gentle shaking. For phospho- STAT1 detection (blue curve), 16 µL of lysate were transferred into a 384-well low volume white microplate, followed by 4 µL of the HTRF phospho-STAT1 detection reagents For total STAT1 detection (red curve), 16µl of lysate were transferred into a 384-well low volume white, followed by 4 µL of the HTRF Total-STAT1 detection reagents. HTRF signals were recorded after an overnight incubation. Note that the HeLa cells display better potency of IFNa compared to NIH3T3 cells.

Detection of phospho and total STAT1 on IFNa stimulated HeLa cells

Detection of phospho and total STAT1 on IFNa stimulated NIH-3T3 cells

Transcriptional activator STAT1 involved in the JAK/STAT pathway

STAT1 is an important transcriptional activator involved in the JAK/STAT pathway which is activated by interferon I class, growth factors or chemokines. After stimulation, phosphorylated STAT1 dimers bind to Interferon Stimulated Gene Factor 3 complex. Then STAT1 proteins translocate into the nucleus and activate the transcription of genes associated to cell survival, viability or pathogen response. In response to IFNγ, STAT1 forms homodimers or heterodimers with STAT3 that bind to the GAS (Interferon-Gamma-Activated Sequence) promoter element. In response to either IFNα or IFNß, STAT1 forms heterodimer with STAT2 that bind the Interferon-Stimulated Response Element (ISRE).

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

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

Open R&D: Sanofi Access Platform

In collaboration with Sanofi - Scientific Presentations

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

HTRF assays for Oncology and Inflammation

Signaling in the Immune System - Brochures

Simplify complex pathway dissection by combining the power of HTRF® cellular phospho-assays and the flexibility of the CyBi®-FeliX liquid handling system

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

HTRF® phospho-protein platform facilitates the dissection of signaling pathways involved in insulin resistance and metabolic disorders

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

Implement HTRF phospho-protein assays at every step of the drug discovery process, from in vitro to in vivo models

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 STAT1 P-Y701 Kit / 63ADK026PEG-63ADK026PEH

63ADK026PEG-63ADK026PEH - Product Insert

HTRF Product Catalog

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

An innate and adaptive immunity recap

Insight into the diversity of cells & signaling pathways - Guides

How to run a cell based phospho HTRF assay

3' video to set up your Phospho assay - Videos

Unleash the potential of your phosphorylation research with HTRF

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

Innovative solutions in Virology research

Kits and reagents for virology research - Flyers

Neuroinflammation study combining hiPSC-derived astrocytes and HTRF

See published experiments and data demonstrating how immunoassays rise to the challenge of astrocyte studies in neuroinflammation research - Application Notes

On-demand webinar: Linking Neuroinflammation and Neurodegeneration

New insight into neuroinflammation research - 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 STAT1 P-Y701 Kit / 63ADK026PEG