Description
The Total STAT3 cellular assay kit is designed to monitor the expression level of STAT3, whether phosphorylated or unphosphorylated. It is compatible with our Phospho-STAT3 kit, and enables the analysis of phosphorylated and total proteins from a single sample for better readout of the JAK/STAT signaling pathway.
HTRF assays offer many advantages over other technologies:
Homogeneous add-and-read format
No wash steps
Low background
Straightforward miniaturization from 96- or 384-well microplates to high density assay formats such as 384-well low volume and 1536-well plates
Stable signal, providing flexibility in time of readout or size of assays
Total-STAT3 assay principle
The Total-STAT3 assay quantifies the expression level of STAT3 in a cell lysate. Contrary to Western Blot, the assay is entirely plate-based and does not require gels, electrophoresis or transfer. The Total-STAT3 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 STAT3 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-STAT3 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 adding Total-STAT3 HTRF detection reagents. This protocol enables the cells' viability and confluence to be monitored.
Total-STAT3 1-plate assay protocol
Detection of total STAT3 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.
HTRF compared to Western Blot using total-STAT3 assay
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.
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.
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 IFNα stimulation, while STAT3 expression level remains constant.
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.
