Total STAT5 assay principle

The Total STAT5 assay quantifies the expression level of STAT5 in a cell lysate. Unlike Western Blot, the assay is entirely plate-based and does not require gels, electrophoresis, or transfer. The Total STAT5 assay uses two labeled antibodies, one coupled to a donor fluorophore and the other to an acceptor. Both antibodies are highly specific for a distinct epitope on the protein. In the presence of STAT5 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 protein's expression under a no-wash assay format.

Total STAT5 two-plate assay protocol

The two-plate protocol involves culturing cells in a 96-well plate before lysis, then transferring lysates into a 384-well low volume detection plate before the addition of HTRF Total STAT5 detection reagents. This protocol allows for the cells' viability and confluence to be monitored.

Total STAT5 one-plate assay protocol

Detection of Total STAT5 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 facilitates miniaturization while maintaining robust HTRF quality.

PROTAC mediated STAT5 degradation in MOLT-4 cells

MOLT-4 cells were dispensed into a 96-well culture plate at a density of 200,000 cells per well and treated with increasing concentrations of the PROTAC AK-2292, known to induce Total STAT5 degradation.

Following overnight incubation, cells were lysed with 10 µL of supplemented lysis buffer #4 (4X) for 30 minutes at room temperature under gentle shaking. Subsequently, 16 µL of lysate was transferred into a 384-well low-volume white microplate, and 4 µL of HTRF total STAT5 detection antibodies were added. Additionally, 4 µL of lysate, supplemented with 12 µL of diluent #8, were transferred into the microplate to assess alpha-tubulin levels using the alpha-tubulin housekeeping Cellular Kit (64ATUBPET/G/H). HTRF signals were recorded after an overnight incubation.

HTRF Phospho-STAT5 (Tyr694) / Total STAT5 modulation using IFN- α on HeLa cells

HeLa cells were plated in a 96-well culture-treated plate at a density of 200,000 cells per well and allowed to adhere in complete culture medium for 24 hours. Subsequently, the cells were exposed to increasing concentrations of IFN-α for 30 minutes at 37°C, 5% CO2, in serum-free cell culture conditions. Following treatment, the cell culture medium was aspirated, and 50 µl of supplemented Lysis Buffer #4 (1X) was added to each well, followed by a 30-minute incubation at room temperature with gentle shaking.

HTRF Phospho-STAT5 (Tyr694) / Total STAT5 modulation using IL-3 on TF-1 cells

TF-1 cells were plated in a 96-well culture plate at a density of 150,000 cells in 25 µL of medium per well and starved in serum-free medium for 2 hours at 37°C, 5% CO2. Subsequently, the cells were exposed to increasing concentrations of IL-3 for 10 minutes in serum-free cell culture conditions.

HTRF Phospho-STAT5 (Tyr694) / Total STAT5 modulation using Lestaurtinib on HeLa cells

HeLa cells were plated in a 96-well culture-treated plate at a density of 200,000 cells per well and incubated for 24 hours in complete culture medium. They were then treated with increasing concentrations of the JAK inhibitor Lestaurtinib for 2 hours, followed by stimulation with 20nM IFN-α for 15 minutes at 37°C, 5% CO2, in serum-free culture medium.

HTRF Phospho-STAT5 (Tyr694) / Total STAT5 modulation using Lestaurtinib on TF-1 cells

TF-1 cells were plated in a 96-well culture plate at a density of 200,000 cells in 20 µL of medium per well and starved in serum-free medium for 2 hours at 37°C, 5% CO2. Following starvation, the cells were treated with increasing concentrations of the JAK inhibitor Lestaurtinib for 2 hours, followed by stimulation with 50nM IL-3 for 10 minutes at 37°C, 5% CO2, in serum-free culture medium.

STAT5 expression level quantified in various cell lines

The suspension cell lines MOLT-4 (T lymphoblast) and Jurkat (T lymphocyte) were dispensed into a 96-well plate and lysed with 10 µL of supplemented lysis buffer #4 (4X) for 30 min at RT under gentle shaking (performed according to the suspension cell protocol).

HTRF Total STAT5 assay compared to Western Blot

MOLT-4 cells were cultured in a T175 flask in complete medium at 37°C, 5% CO2. After medium removal, the cells were lysed with 3 mL of supplemented lysis buffer #4 (1X) for 30 min at RT under gentle shaking.

STAT5 signaling pathway

The activation of STAT5 proteins, is mediated by cytokines, and other growth factors, allowing rapid delivery of signals from the membrane to the nucleus. These STATs regulate the expression of target genes in a cytokine-specific fashion. In addition to their activation by cytokines, the activities of Stat5a and Stat5b are negatively controlled by CIS/SOCS/SSI proteins.

JAK/STAT pathway activation requires ligand binding to the receptor, which in consequence activates the Janus kinases JAKs, a family of intracellular tyrosine kinases. Via phosphorylation of downstream signal transducers and activators of transcription (STAT), STAT5 form homo or heterodimers (STAT5a and STAT5b) and translocate into the nucleus, where they bind to STAT5 response elements, inducing the transcription of specific sets of genes.