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HTRF Human and Mouse Total STAT6 Detection Kit HTRF®

This HTRF kit detects cellular STAT6 and can be used as a normalization assay with our phospho-STAT6 kit for an optimal readout of JAK/STAT signaling.

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  • No-wash No-wash
  • All inclusive kit All inclusive kit
  • Low sample consumption Low sample consumption
  • High sensitivity High sensitivity

This HTRF kit detects cellular STAT6 and can be used as a normalization assay with our phospho-STAT6 kit for an optimal readout of JAK/STAT signaling.

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Overview

The Total STAT6 cellular assay kit is designed to monitor the expression level of STAT6, whether phosphorylated or unphosphorylated. It is compatible with our Phospho-STAT6 kit, and enables the analysis of phosphorylated and total proteins from a single sample for better readouts of the JAK/STAT signaling pathway.

Benefits

  • SPECIFICITY
  • PRECISION

Total STAT6 assay principle

The Total STAT6 assay quantifies the expression level of STAT6 in a cell lysate. Unlike Western Blot, the assay is entirely plate-based and does not require gels, electrophoresis, or transfer. The Total-STAT6 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 STAT6 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.

Principle of the HTRF total STAT6 assay

Total STAT6 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 the addition of Total-STAT6 HTRF detection reagents. This protocol enables the cells' viability and confluence to be monitored.

2-plate protocol of the HTRF total STAT6 assay

Total STAT6 1-plate assay protocol

Detection of Total-STAT6 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.

1-plate protocol of the HTRF total  STAT6 assay

Total STAT6 detection on human and mouse cell lines

HeLa, Raw264.7 (mouse) cells, or suspensions such as THP1 cells were seeded at 100,000 cells / well in a 96-well microplate. After a 24H incubation, the cells were lysed with supplemented lysis buffer, and 16 µL of lysate were transferred into a 384-well low volume white microplate before the addition of 4 µL of the HTRF Total STAT6 detection reagents. The HTRF signal was recorded after an overnight incubation.

The HTRF Total STAT6 assay efficiently detected STAT6 in various cellular models expressing different levels of the protein.

Total STAT6 detection on various cell lines

Specificity of HTRF Total STAT6 assay using knockout HAP1 cell lines

STAT6 expression level was assessed with the HTRF total STAT6 kit in HAP1 cells (WT) and the HAP1 cell line knocked-out for STAT6. Cells were seeded at 200,000, 100,000, 50,000, and 25,000 cells/ well in a 96-well microplate and cultured for 24 hours at 37°C, 5% CO2. After culture medium removal, the cells were lysed with 50 µL of supplemented lysis buffer #4 (1X). Then 16 µL of cell lysate were transferred into a low volume white microplate, followed by 4 µL of premixed detection reagents. The HTRF signal was recorded after an overnight incubation at RT.

In HAP1 KO STAT6 cells, the HTRF signal was equivalent to the non-specific signal (dotted line), indicating complete STAT6 gene silencing and good assay specificity, whereas the STAT6 level was well detected in the wild-type cells, as expected.

Specificity of Total STAT6 assay using knock out HAP1 cell line

Stimulation of phospho STAT6 Tyr641 in HeLa cell lines

HeLa cells were seeded in a 96-well culture-treated plate under 50,000 cells / well in complete culture medium, and incubated overnight at 37 ° C, 5% CO2. Cells were then stimulated with increasing concentrations of hIL4 for 20 minutes. Following the 2-plate assay protocol, 16 µL of lysate were transferred into a 384-well low volume white microplate before the addition of 4 µL of the HTRF phospho-STAT6 (Tyr641) #64AT6PEG/H/Y or Total STAT6 detection reagents. The HTRF signal was recorded after an overnight incubation.

As expected, the results obtained showed a dose-response stimulation of STAT6(Tyr641) phosphorylation upon treatment with hIL4, while the STAT6 expression level remained constant.

Stimulation of phospho STAT6 Tyr641 in HeLa cell lines

Inhibition of phospho STAT6 Tyr 641 in HeLa cell line

HeLa cells were seeded in a 96-well culture-treated plate under 50,000 cells / well in complete culture medium, and incubated overnight at 37 ° C, 5% CO2. The cells were then treated with increasing concentrations of Ruxolitinib or JAK inhibitor 1 for 2H at 37°C, 5% CO2, followed by stimulation with hIL4 at 5 ng/mL for 20 minutes.

After cell lysis, 16 µL of lysate were transferred into a 384-well sv white microplate, and 4 µL of the HTRF phospho-STAT6 (Tyr641) or Total STAT6 detection reagents were added. The HTRF signal was recorded after an overnight incubation at room temperature.

As expected, the results obtained showed a dose-response inhibition of STAT6 Tyr641 phosphorylation upon treatment with Ruxolitinib or JAK inhibitor 1, while the STAT6 expression level remained constant.

Inhibition of phospho STAT6 Tyr 641 in HeLa cell line
Inhibition of phospho STAT6 Tyr 641 in HeLa cell line

HTRF total STAT6 assay compared to Western Blot

HeLa cells were cultured in a T175 flask in complete culture medium at 37°C, 5% CO2. After a 48h incubation, the cells were lysed with 3 mL of supplemented lysis buffer #4 (1X) for 30 minutes at RT under gentle shaking.

Serial dilutions of the cell lysate were performed using supplemented lysis buffer, and 16 µL of each dilution were transferred into a low volume white microplate before the addition of 4 µL of HTRF total STAT6 detection reagents. Equal amounts of lysates were used for a side by side comparison between HTRF and Western Blot.

Using the HTRF total STAT6 assay, 300 cells/well were enough to detect a significant signal, while 20,000 cells were needed using Western Blot with an ECL detection. Therefore in these conditions, the HTRF total STAT6 assay is 60 times more sensitive than the Western Blot technique.

Comparison between HTRF and WB sensitivity on Total STAT6

Function and regulation of STAT6

STAT6 is phosphorylated in response to cytokines and growth factors by the receptor associated kinase, JAK. Once phosphorylated, STAT6 proteins form homo or heterodimers and translocate into the nucleus, where they mediate cytokine induced gene expression. Interleukin 4 is the main cytokine triggering STAT6 phosphorylation on tyrosine 641 residue and inducing the expression of BCL2L1/BCL-X(L), which is responsible for the anti-apoptotic activity of IL4. To a lesser extent, STAT6 phosphorylation by TBK1 has been shown to be associated with the STING pathway, thus playing a role in innate immunity in response to viral infection.

Total STAT6 signaling pathway

HTRF phospho-assays reveal subtle drug-induced effects

Detailed protocol and direct comparison with WB - Posters

HTRF® cell signaling platform combined with iCell® Hepatocytes

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

Key guidelines to successful cell signaling experiments

Mastering the art of cell signaling assays optimization - Guides

Optimize your HTRF cell signaling assays on tissues

HTRF and WB compatible guidelines - Technical Notes

HTRF Alpha-tubulin Housekeeping kit

Properly interpret your compound effect - Application Notes

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

Insider Tips for successful sample treatment - Technical Notes

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

Increased flexibility of phospho-assays - Application Notes

HTRF cell-based phospho-protein data normalization

Valuable guidelines for efficiently analyzing and interpreting results - Application Notes

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

Tumor xenograft analysis: HTRF versus Western blot - Application Notes

Best practices for analyzing tumor xenografts with HTRF phospho assays

Protocol for tumor xenograft analysis with HTRF - Technical Notes

Unleash the potential of your phosphorylation research with HTRF

Unmatched ease of use, sensitivity and specificity assays - Videos

How to run a cell based phospho HTRF assay

What to expect at the bench - Videos

TCR signaling investigation with HTRF phospho assays

Study a pathway of interest in PBMC and T cells - Application Notes

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

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

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

Investigating kinase activity in a cellular context

HTRF cellular assays - Scientific Presentations

Species compatibility

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

HTRF cellular phospho-protein assays

Physiologically relevant results fo fast flowing research - Flyers

Lysis buffer compatibility

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

Open R&D: Sanofi Access Platform

In collaboration with Sanofi - Scientific Presentations

Biomarker and Cell Signaling Assays for Fibrosis and NASH

HTRF and Alpha solutions for NASH - Flyers

Inflammation cell by cell

HTRF solutions for each cell type - Flyers

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

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

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

Manual STAT6 total Kit / 64stat6tpeg-64stat6tpeh

64stat6tpeg-64stat6tpeh - Product Insert

Certificate of Analysis STAT6 total Kit / 64STAT6TPEG

64STAT6TPEG Batch 01B - Quality Control Report

Certificate of Analysis STAT6 total Kit / 64STAT6TPEG

64STAT6TPEG Batch 01C - Quality Control Report

Certificate of Analysis STAT6 total Kit / 64STAT6TPEG

64STAT6TPEG Batch 01D - Quality Control Report

Certificate of Analysis STAT6 total Kit / 64STAT6TPEH

64STAT6TPEH Batch 01A - Quality Control Report

Plate Reader Requirement

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