Based on our homogeneous and robust HTRF assays, the Phospho-STAT3 kit is designed for detecting and studying activated STAT3 when phosphorylated at Tyr705 directly in whole cells. Using a streamlined protocol, amenable to low-volume format, this kit can be used from basic research to High Throughput drug screening.
The assay can be run with cell lysates or using whole cells.
Upon activation, STAT3 is phosphorylated on Tyr705 and after the lysis of the cell membrane, phospho- STAT3 can be detected using the kit reagents. The assays are based on sandwich immunoassays, each involving two monoclonal antibodies: the anti-phospho-STAT3 antibody labeled with Eu3+-cryptate and the anti-STAT3 antibody labeled with d2. These antibodies may be pre-mixed and added in a single dispensing step to further streamline the protocol.
Cells are plated, stimulated and next lysed in the same 96-well culture plate. Lysates are then transferred to the assay plate for the detection of phosphorylated STAT3 by HTRF reagents. This protocol enables the cells' viability and confluence to be monitored.
Detection of phosphorylated STAT3 with HTRF reagents is performed in a single plate used for plating, stimulation and lysis. No washing steps are required. This protocol, HTS designed, enables miniaturization while maintaining HTRF quality.
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 others receptors such as RTKs (EGFR, cmet.) and c-src.
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.
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.
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.
| Description | Cat. no | Product insert | MSDS |
|---|---|---|---|
| Phospho STAT3 Assay kit - 500 tests | 62AT3PEG | - | |
| Phospho STAT3 Assay kit - 10,000 tests | 62AT3PEH | - | |
| Phospho STAT3 Assay kit - 50,000 tests | 62AT3PEI | - |
| Description | Cat. no | Product insert | MSDS |
|---|---|---|---|
| Phospho STAT3 control lysate | 62AT3TDA | - | - |
| Cellular kinase blocking reagent - 2 ml | 64KB1AAC | - | |
| Cellular kinase blocking reagent - 6 ml | 64KB1AAD | - | |
| Cellular kinase lysis buffer - 130 mL | 64KL1FDF | - | |
| Total STAT3 Assay kit - 500 tests | 64NT3PEG | ||
| Total STAT3 Assay kit - 10,000 tests | 64NT3PEH | ||
| Total STAT3 Assay kit - 50,000 tests | 64NT3PEI |
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