Phospho-STAT3 (Tyr705) & Total STAT3 Cellular Assay Kits

HTRF® cell-based phospho and total-STAT3 assays for anti-cancer research

In response to cytokines and growth factors, STAT3, Signal transducer and activator of transcription 3 is phosphorylated at Tyr705 by activation of JAK proteins, or by various receptor tyrosine kinases. STAT3 has been found to be upregulated in many cancers including glioblastoma, head and neck cancer, prostate cancer, and breast cancer. A constitutively active form of STAT3 is oncogenic. STAT 3 activation is associated with Crohn's disease, and other inflammatory diseases such as pulmonary fibrosis and acute lung injury. STAT3 is critical for leptin signaling and its mutation leads to obesity in mice.

Cisbio’s homogeneous and robust HTRF® phospho-STAT3 (Tyr705) assay kit is designed for the cell-based quantitative detection of STAT3 signaling activity. The HTRF® total STAT3 assay kit is designed for the quantitative detection of total STAT3, phosphorylated and unphosphorylated, to normalize the phosphorylation status of STAT3 with respect to its steady-state level in cells.

The buffers of both HTRF® phospho- and total STAT3 assays are compatible, enabling an analysis of the phosphorylated and the total protein populations from one lysate sample.

   Check our lysis buffer compatibility      Check our species compatibility

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Assay principle

Cisbio Bioassay’s phospho- and total STAT3 assays are based on a TR-FRET sandwich immunoassay format comprising two specific anti-STAT3 antibodies, one labeled with Cryptate (donor) and the other with d2 (acceptor). The antibodies specifically bind with STAT3, and the proximity of donor and acceptor then leads to a fluorescent TR-FRET signal. 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 assays can be run with frozen cell lysates or fresh cells in culture. After cell lysis, endogenous phospho- or total-STAT3 can be quantitatively detected using the HTRF phospho-STAT3 (Tyr705) and total STAT3 cellular assay kit reagents and most TR-FRET multimode plate readers.

A simpler, more flexible assay protocol – adapted to your applications

Two-plates assay protocol

For added flexibility, the assay can be run under a two-plate assay protocol, where cells are plated and treated in a 96-well culture plate. For detection, lysates are subsequently transferred to a 384-well small volume assay plate where the HTRF reagents are added. This also enables monitoring the cells' viability and confluence in an appropriate cell culture plate.

One-plate assay protocol

The protocol can be further streamlined to a one-plate assay in which plating, treating and detecting are all performed in a single plate. No wash steps are required. Ideal for HTS - this protocol means enhanced speed and simplicity, enabling all throughputs and fast results while maintaining high quality and sensitive output.

What to expect at the bench

This video covers the principles of phospho-HTRF assays and gives guidelines for performing them, using our phospho-ERK assay as an example.

Simplified Pathway

STAT3 Epidermal growth factor signaling

In response to cytokines and growth factors, STAT3 is phosphorylated by receptor-associated kinases and then forms dimers that translocate to the cell nucleus, where they act as transcription activators. STAT3 mediates the expression of a variety of genes in response to cell stimuli, and thus plays a key role in many cellular processes such as cell growth and apoptosis. The binding of Interleukin 6—family cytokines to the gp130 receptor triggers STAT3 phosphorylation by JAK2. STAT3 is also a target of other receptors such as RTKs (EGFR, cmet.) and c-src

Product Performances

1. HTRF Phospho-STAT3 assay compared to Western Blot

HeLa cells were grown in a T175 flask 37°C, 5%Co2, 2days.Stimulation was done with IFNα 5nM for 15min. After elimination of cell culture medium, 3ml of supplemented lysis buffer was added and incubated for 45min. Soluble supernatants were collected after 10min centrifuging. Equal amounts of lysates were used for a side by side comparison of WB and HTRF.

The two methods show a comparable sensitivity level: 750 cells. HTRF offers superior convenience over western blot.

2. HTRF total-STAT3 assay compared to Western Blot

HEK293 cells were grown in a T175 flask at 37°C, 5% CO2 for 2 days. After removal of cell culture medium, 3mL of supplemented lysis buffer were added and incubated for 45 min. Soluble supernatants were collected after 10 min centrifuging. Equal amounts of lysates were used for a side by side comparison of WB and HTRF. 150,000 cells correspond to approximately 20µg of total protein. The two methods show a comparable sensitivity level: 1172 cells could be detected with each technology.

3. IL6 dose-response on NIH 3T3 cells

Murine NIH 3T3 cells (100,000 cells/well) were stimulated for 30 minutes at 37°C with various concentrations of IL6. After a 30-minute lysis incubation time, phosphorylated STAT3 was measured using the two-plate assay protocol of the HTRF phopsho-STAT3(Tyr705) cellular assay kit.

4. Inhibition effect of JAK inhibitor on HeLa cells

HeLa cells (100,000 cells/well) were incubated for 1 hour at 37°C with various concentrations of antagonist. Agonist (IFNα) was then added and incubated for 30 minutes. After a 30-minute lysis incubation time, phosphorylated STAT3 was measured using the two-plate assay protocol of the HTRF phopsho-STAT3(Tyr705) cellular assay kit.

5. Validation of the total-STAT3 kit on various species of cell lines: human, mouse and hamster

Human (Hela, A431, HEK293, Jurkat), murine (NIH 3T3) and Hamster (CHO-K1) cells were grown in a T175 flask at 37°C, 5% CO2 for 2 days. After removal of cell culture medium, 3mL of supplemented lysis buffer 3 were added and incubated for 45 min. Soluble supernatants were collected after 10 min centrifuging. STAT3 was detected using 16µL of cell lysate.

6. HTRF® total-STAT3 assay used to check the phosphorylation status of STAT3

 

 

Hela cells (100,000 cells/well) were activated with IFNa for 15 min, using the two-plate assay protocol of the Phospho-STAT3 (Tyr705) and Total-STAT3 assays.

Results obtained show a dose-response increase of STAT3 phosphorylation upon IFNa stimulation, while STAT3 expression level remains constant.

Part#, inserts & MSDS

Ordering Info

DescriptionCat. noProduct insertMSDS
STAT3 phospho-Y705 kit - 500 tests62AT3PEG
STAT3 phospho-Y705 kit - 10,000 tests62AT3PEH
STAT3 phospho-Y705 kit - 1 x 96 tests62AT3PET
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STAT3 phospho-Y705 kit - 50,000 tests62AT3PEY
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STAT3 phospho-Y705 kit control lysate62AT3TDA
STAT3 total kit - 500 tests64NT3PEG
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STAT3 total kit - 10,000 tests64NT3PEH
-
STAT3 total kit - 50,000 tests64NT3PEY
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Companion products

DescriptionCat. noProduct insertMSDS
STAT3 total kit control lysate64NT3TDA
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