Description
The total STAT1 assay measures endogeneous levels of STAT1 in cell lysates. STAT1 acts as an important transcriptional activator and the assay serves as a readout for JAK inhibitors in oncology, virology, and inflammation. The assay can be used to normalize the amount of phosphorylated STAT1 over total STAT1.
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-STAT1 assay principle
The Total-STAT1 assay quantifies the expression level of STAT1 in a cell lysate. Contrary to Western Blot, the assay is entirely plate-based and does not require gels, electrophoresis or transfer. The Total-STAT1 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 STAT1 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-STAT1 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-STAT1 HTRF detection reagents. This protocol enables the cells' viability and confluence to be monitored.
Total-STAT1 1-plate assay protocol
Detection of total STAT1 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 assay compared to Western Blot using total STAT1 cellular assays on human jurkat cells
HeLa cells were grown in a T175 flask at 37 °C, 5% CO2 for 48h. Cells were then stimulated with IFNa 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.
HTRF total-STAT1 assay used to check the phosphorylation status of STAT1 on murine and human cells
After 20 minutes of stimulation with increasing IFNa concentrations, NIH 3T3 & Hela cells (200,000 cells/96well 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 IFNa compared to NIH3T3 cells.
Transcriptional activator STAT1 involved in the JAK/STAT 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 Interferon Stimulated Gene Factor 3 complex. Then STAT1 proteins translocate into the nucleus and activate the transcription of genes associated to 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 IFNß, STAT1 forms heterodimer with STAT2 that bind the Interferon-Stimulated Response Element (ISRE).
