STAT1 phospho (Tyr701) & total assays

HTRF® cell-based immunoassay for quantification of total and phospho-STAT1 in JAK/STAT signaling

Cisbio's cell-based homogeneous HTRF® phospho-STAT1 assay (Tyr701) is designed for the quantitative detection of STAT1 modulation phosphorylated on tyrosine 701, while the HTRF® total STAT1 assay is designed for the quantitative detection of STAT1 modulation. Stimulated by growth factors and chemokines, like IFN-γ induced JAK/STAT signaling, STAT1 acts as an important transcriptional activator. Thus the assay serves as a readout for JAK inhibitors in oncology, infectiology, and inflammation.

The phospho-STAT1 and total STAT1 ready-to-use kits contain all the reagents you need and offer enhanced convenience over other immunoassay technologies, being easily amenable to high throughput screening and automated platforms.

   Check our lysis buffer compatibility      Check our species compatibility

Pathway

HTRF® - the homogeneous cell-based sandwich immunoassay

Total and phospho-STAT1 Tyr701 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 the Interferon Stimulated Gene Factor 3 complex. Then STAT1 proteins translocate into the nucleus and activate the transcription of genes associated with 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 IFNβ, STAT1 forms heterodimers with STAT2 that bind the Interferon-Stimulated Response Element (ISRE).

Assay principle

HTRF® - the homogeneous cell-based sandwich immunoassay

The phospho-STAT1 assay is based on a TR-FRET sandwich immunoassay format comprising two specific anti-STAT1 antibodies, one labeled with a cryptate as donor and the other with d2 as acceptor. The phospho-STAT1 antibodies bind the phosphorylated residue, and the proximity of donor and acceptor will then lead to a fluorescent TR-FRET signal. The signal intensity is proportional to the substrate phosphorylation. The protocol is optimized for a 384-well plate format, but can easily be further miniaturized or upscaled. Only low sample volumes are needed. The detection reagents may be pre-mixed and added in a single dispensing step for direct detection. No washing is needed at any step.

The total or phospho-STAT1 assay kits can be run with frozen cell lysates or fresh cells in culture. After cell lysis, total or phospho-STAT1 can be quantitatively detected using the HTRF total or phospho-STAT1 kit reagents and most TR-FRET multimode plate readers.

phospho total pathway

Product performances

1. Detection of phospho STAT1 on HeLa cells: stimulation kinetic

IFNα was used to induce phosphorylation of STAT1 in HeLa cells. 200,000 cells were plated in a 96-well plate, then stimulated with increasing IFNα 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 with IFNa .

Phospho stat1 kinetic stimulation

2. 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 displays the biological assay window corresponding to IFNa-stimulated over unstimulated conditions.

Phospho stat1 y701 cell densities

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

After a 20 minute stimulation with increasing IFNa concentrations, NIH 3T3 & HeLa cells (200,000 cells/96-well 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 IFNalpha  compared to NIH3T3 cells.

Phospho stat1 IFNalpha stimulation hela cells

Phospho stat1 ifn alpha stimulation nih 3t3 cells

4. HTRF phospho-STAT1 Y701 assay compared to western blot

HeLa cells were grown in a T175 flask at 37 °C, 5% CO2 for 48h. Cells were then stimulated with IFNalpha 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.

phospho stat1 y701 hela cell lysate dilutions

5. HTRF total-STAT1 assay compared to western blot

HeLa cells were grown in a T175 flask at 37 °C, 5% CO2 for 48h. Cells were then stimulated with IFNalpha 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.

phospho stat1 hela cell lysate dilutions

Part#, inserts & MSDS

Ordering Info

DescriptionCat. noProduct insertMSDS
STAT1 phospho-Y701 kit - 500 tests63ADK026PEG
STAT1 phospho-Y701 kit - 10,000 tests63ADK026PEH
STAT1 phospho-Y701 kit - 50,000 tests63ADK026PEY
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STAT1 total kit - 500 tests63ADK096PEG
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STAT1 total kit - 10,000 tests63ADK096PEH
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STAT1 total kit - 50,000 tests63ADK096PEY
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Companion products

DescriptionCat. noProduct insertMSDS
STAT1 phospho-Y701 kit - control lysate63ADK026TDA
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STAT1 total kit control lysate63ADK096TDA
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