Description
STAT5, comprising STAT5A and STAT5B, is activated by JAK kinases in response to cytokine signaling. Upon phosphorylation, STAT5 forms dimers and translocates to the nucleus, inducing transcription of genes involved in proliferation, differentiation, and survival. Overactive STAT5 drives cancers like leukemia and breast cancer, while its dysregulation contributes to autoimmune diseases by promoting excessive immune responses.
The AlphaLISA SureFire Biotin-Free Human and Mouse Total STAT5 Detection Kit is a sandwich immunoassay for the quantitative detection of total STAT5 in cellular lysates, using Alpha Technology.
Formats:
The HV (high volume) kit contains reagents to run 100 wells in 96-well format, using a 60 μL reaction volume.
The 500-point kit contains enough reagents to run 500 wells in 384-well format, using a 20 μL reaction volume.
The 10,000-point kit contains enough reagents to run 10,000 wells in 384-well format, using a 20 μL reaction volume.
The 50,000-point kit contains enough reagents to run 50,000 wells in 384-well format, using a 20 μL reaction volume.
AlphaLISA SureFire Biotin Free kits are compatible with:
Cell and tissue lysates
Antibody modulators
Biotherapeutic antibodies
Biotin rich samples
AlphaLISA SureFire Biotin Free kits can be used for:
Cellular kinase assays
Receptor activation studies
High-throughput screening for preclinical studies
Total-AlphaLISA SureFire Biotin Free assay principle
The Total-AlphaLISA SureFire Biotin Free assay measures the expression level of a target protein in a biological sample (e.g. cell lysate).
The Total-AlphaLISA SureFire Biotin Free assay uses two antibodies which recognize two different distal epitopes on the target protein. AlphaLISA assays require two bead types: Acceptor and Donor Beads. Acceptor Beads are coated with a proprietary CaptSure™ agent to specifically immobilize the assay specific antibody, labeled with a CaptSure tag. Donor Beads are coated with a proprietary CaptSure3 agent to capture one of the detection antibodies, which is labeled with the CaptSure3 tag. In the presence of target protein, the two antibodies bring the Donor and Acceptor Beads in close proximity whereby the singlet oxygen transfers energy to excite the Acceptor Bead, allowing for the generation of a luminescent Alpha signal. The amount of light emission is directly proportional to the quantity of protein present in the sample.
Total-AlphaLISA SureFire Biotin Free two-plate assay protocol
The two-plate protocol involves culturing and treating the cells in a 96-well plate before lysis, then transferring lysates into a 384-well OptiPlate™ plate before the addition of Total-AlphaLISA SureFire Biotin Free detection reagents. This protocol enables cell viability and confluence to be monitored. In addition, lysates from a single well can be used to measure multiple targets.
Total-AlphaLISA SureFire Biotin Free one-plate assay protocol
Detection of Total target protein with AlphaLISA SureFire Biotin Free reagents can be performed in a single plate used for culturing, treatment, and lysis. No washing steps are required. This HTS designed protocol allows for miniaturization while maintaining robust AlphaLISA SureFire quality.
Validation of Phospho (Tyr694/699)/Total STAT5 levels in IFNα or IFNγ treated cells
THP-1 cells were seeded in a 96-well plate (50,000 cells/well) in complete RPMI 1640 medium containing 100 nM of PMA for 24 hours at 37°C, 5% CO2. The THP-1 derived macrophages were starved for 2 hours and then treated with increasing concentrations of human interferon alpha (IFNα) or human interferon gamma (IFNγ) for 20 minutes.
After treatment, the cells were lysed with the addition of 50 µL of 5X Lysis Buffer for 10 minutes at RT with shaking (350 rpm). STAT5 Phospho (Tyr694/699) and Total levels were evaluated using respective AlphaLISA SureFire Biotin Free assays. For the detection step, the lysate was further diluted 1:2 with Lysis Buffer, 10 µL of cell lysate (approximately 1,000 cells) were transferred into a 384-well white OptiPlate, followed by 5 µL of Acceptor mix and incubated for 1 hour at RT. Finally, 5 µL of Donor mix was then added to each well and incubated for 1 hour at RT in the dark. The plate was read on an Envision using standard AlphaLISA settings.
As expected, IFNα or IFNγ triggered a dose-dependent increase in the levels of Phospho STAT5 (Tyr694/699) while Total STAT5 levels remained unchanged.
Degradation of endogenous Total STAT5 in MOLT-4 cells
MOLT-4 cells were seeded in a 96-well plate (200,000 cells/well) in complete RPMI 1640 medium and treated with increasing concentrations of STAT5 PROTAC (AK-2292) for 20 hours at 37°C, 5% CO2.
After treatment, the cells were lysed with the addition of 50 µL of 5X Lysis Buffer for 10 minutes at RT with shaking (350 rpm). STAT5 Total and STAT3 Total levels were evaluated using respective AlphaLISA SureFire Biotin Free assays. For the detection step, 10 µL of cell lysate (approximately 8,000 cells) were transferred into a 384-well white OptiPlate, followed by 5 µL of Acceptor mix and incubated for 1 hour at RT. Finally, 5 µL of Donor mix was then added to each well and incubated for 1 hour at RT in the dark. The plate was read on an Envision using standard AlphaLISA settings.
As expected, AK-2292 triggered a dose-dependent decrease in the levels of STAT5 (DC50 = 0.52 µM), while Total STAT3 levels were unchanged.
