This HTRF kit detects cellular STAT1 and can be used as a normalization assay with our phospho-STAT1 kit for an optimal readout of JAK/STAT signaling.

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  • 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 STAT1 and can be used as a normalization assay with our phospho-STAT1 kit for an optimal readout of JAK/STAT signaling.

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Overview

The total STAT1 assay measures endogeneous levels of STAT1 in cell lysates. STAT1 acts as an important transcriptional activator and the assay serves as a readout for JAK inhibitors in oncology, virology, and inflammation. The assay can be used to normalize the amount of phosphorylated STAT1 over total STAT1.

Benefits

  • SPECIFICITY
  • PRECISION
  • DATA NORMALIZATION

Total-STAT1 assay principle

The Total-STAT1 assay quantifies the expression level of STAT1 in a cell lysate. Contrary to Western Blot, the assay is entirely plate-based and does not require gels, electrophoresis or transfer. The Total-STAT1 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 STAT1 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-STAT1 assay principle

Total-STAT1 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-STAT1 HTRF detection reagents. This protocol enables the cells' viability and confluence to be monitored.
Total-STAT1 2-plate assay protocol

Total-STAT1 1-plate assay protocol

Detection of total STAT1 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-STAT1 1-plate assay protocol

HTRF assay compared to Western Blot using total STAT1 cellular assays on human Jurkat 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 total STAT1.
HTRF assay compared to Western Blot using total STAT1 cellular assays on human Jurkat cells

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

After 20 minutes of 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).

Regulation pathway of STAT1

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

HTRF assays for Oncology and Inflammation

Signaling in the immune system - Brochures

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 STAT1 total Kit / 63ADK096PEG-63ADK096PEH

63ADK096PEG-63ADK096PEH - Product Insert

Cisbio Product Catalog 2019

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

An innate and adaptive immunity recap

Insight into the diversity of cells & signaling pathways - Guides

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