Phospho-STAT6 (Tyr641) Cellular Kit

This cell-based assay is designed to monitor the phosphorylation of STAT6 on Tyr641, which represents a hallmark of its activation. 

STAT6 is a transcription factor mainly activated by the IL4 receptor, and also by the IL3, IL13 or IFNa receptor. STAT6 is phosphorylated on tyrosine 641 by Janus kinases, which leads to the formation of homodimers or heterodimers with STAT2. Finally, activated STAT6 translocates to the nucleus and mediates cytokine-induced gene expression.

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Assay principle

Phospho-STAT6 (Tyr641) assay principle

The Phospho-STAT6 (Tyr641) assay measures STAT6 when phosphorylated at Tyr641. Unlike Western Blot, the assay is entirely plate-based and does not require gels, electrophoresis, or transfer. The Phospho-STAT6 (Tyr641) assay uses 2 labeled antibodies, one with a donor fluorophore and the other 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 independently of its phosphorylation state. Protein phosphorylation enables an immune-complex formation involving both labeled antibodies 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 protein’s phosphorylation state under a no-wash assay format.

Phospho STAT6 Y641 assay principle

Phospho-STAT6 (Tyr641) 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 Phospho-STAT6 (Tyr641) HTRF detection reagents. This protocol enables the cells' viability and confluence to be monitored.

Phospho-STAT6 (Tyr641) 1-plate assay protocol

Phospho-STAT6 (Tyr641) 1-plate assay protocol

Detection of Phosphorylated STAT6 (Tyr641) 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-STAT6 (Tyr641) 1-plate assay protocol

Assay Validation

1. STAT6 Tyr641 phosphorylation assessed on various cell models

Various human cell lines, either adherent HeLa cells or suspensions such as TF1, U937 and TH1 cells, were seeded at 100,000 cells / well in a 96-well microplate, then stimulated with 5ng/mL of hIL4 for 20 minutes. After cell lysis with 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-STAT6 (Tyr641) detection reagents. The HTRF signal was recorded after an overnight incubation.

Image of the validation of the Cisbio HTRF Phospho STAT6 on the adherent and suspension cell lines treated with IL4

2. Pharmacological validation of phospho-STAT6 (Tyr641) in THP1 cells

THP1 cells were seeded at different cell densities in 96-well microplates, 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) detection reagents. The HTRF signal was recorded after an overnight incubation.

 

3. STAT6 phosphorylation induced by IFNα dose-response

THP1 cells were seeded at different cell densities in a 96-well microplate, then stimulated with increasing concentrations of hIFNa 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) detection reagents. The HTRF signal was recorded after an overnight incubation.

Image of the comparison between Western blot and HTRF using phospho-STAT6 on human HeLa cells treated with IL4

4. HTRF phospho-STAT6 cellular assay compared to Western Blot

The human Hela line was seeded in a T175 flask, and incubated at 37°C, 5% CO2, until confluency. The cells were then treated with IL4 (50 ng/mL) for 20 min before lysis.

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

Using the HTRF Phospho-STAT6 Y641 assay, 5,000 cells/well were sufficient to detect a signal, while 40,000 cells were needed using Western Blot with an ECL detection. These results demonstrate that the HTRF phospho-STAT6 assay is 8 times more sensitive than the Western Blot.

Simplified pathway

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.

STAT6 signaling pathway

Part#, inserts & MSDS

Ordering Info

DescriptionCat. noProduct insertMSDS
STAT6 phospho-Y641 kit - 500 tests64AT6PEG
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STAT6 phospho-Y641 kit - 10,000 tests64AT6PEH
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STAT6 phospho-Y641 kit - 50,000 tests64AT6PEY
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Companion products

DescriptionCat. noProduct insertMSDS
STAT6 phospho-Y641 kit - control lysate64AT6TDA
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