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 proteins expression under a no-wash assay format.

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.

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.

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.

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.

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.

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.

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.