Description
Overview Hypoxia-Inducible Factor 2-Alpha (HIF2α) is an oxygen-sensitive transcription factor that regulates cellular adaptation to hypoxia by controlling genes involved in angiogenesis, erythropoiesis, and metabolism. Under normoxic conditions, HIF2α is hydroxylated by prolyl hydroxylases, leading to VHL-mediated degradation. During hypoxia, HIF2α stabilizes and heterodimerizes with HIF1β to bind hypoxia response elements in target gene promoters. HIF2α uniquely regulates erythropoietin, VEGF in endothelial cells, and genes involved in lipid metabolism. Mutations in VHL or HIF2α cause polycythemia and clear cell renal cell carcinoma, where constitutive HIF2α activity drives tumor growth. HIF2α-specific inhibitors such as belzutifan have shown remarkable efficacy in ccRCC. The AlphaLISA SureFire Ultra Human Total HIF2α is a sandwich immunoassay for the quantitative detection of total HIF2α 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 Ultra kits are compatible with: Cell and tissue lysates Antibody modulators Biotherapeutic antibodies AlphaLISA SureFire Ultra kits can be used for: Cellular kinase assays Receptor activation studies High-throughput screening for preclinical studies How it works Total-AlphaLISA SureFire Ultra assay principle The Total-AlphaLISA SureFire Ultra assay measures the expression level of a protein target in a cell lysate. The Total-AlphaLISA SureFire Ultra assay uses two antibodies which recognize two different distal epitopes on the targeted 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 streptavidin to capture one of the detection antibodies, which is biotinylated. In the presence of targeted 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 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 Ultra 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 Ultra detection reagents. This protocol permits the cells viability and confluence to be monitored. In addition, lysates from a single well can be used to measure multiple targets. Total-AlphaLISA SureFire Ultra one-plate assay protocol Detection of Total target protein with AlphaLISA SureFire Ultra 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 AlphaLISA SureFire Ultra quality. Assay versatility HIF-2α expression in various cell lines Adherent cells were seeded at 40,000 cells/well in a 96-well culture plate in complete medium and incubated overnight at 37°C, 5% CO2 . Cells were treated with CoCl2 for 4 hours, then washed with HBSS and lysed with 100 µL of lysis buffer. HIF-2α total levels were evaluated by AlphaLISA SureFire Ultra. For the detection step, 10 µL of cell lysate (approximately 4,000 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. Assay sensitivity Assay sensitivity - cell lysate Cell lysate was prepared from HEK293 cells cultured to confluence a T175 flask and stimulated with 250 µM CoCl2 for 18 hours. Cells were lysed with Lysis Buffer for 10 minutes at RT with shaking. Lysate was serially diluted in Lysis Buffer and HIF-2α Total levels were evaluated by AlphaLISA SureFire Ultra . For the detection step, 10 µL of lysate was transferred into a 384-well white OptiPlate, followed by 5 µL of Acceptor mix and incubated for 1 hour at room temperature. 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. Approximate number of cells per datapoint is indicated. The dotted line represents assay background. The assay can detect HIF-2α Total down to 500 cells/datapoint.
