Description
Overview Paxillin is a focal adhesion adaptor protein that serves as a scaffolding platform for integrating signals from integrins, growth factor receptors, and cytoskeletal components. It localizes to focal adhesions where it coordinates cell adhesion, migration, and survival through interactions with kinases (FAK, Src), structural proteins (vinculin, talin), and signaling molecules (Crk, p120RasGAP). Paxillin undergoes phosphorylation at multiple tyrosine and serine residues in response to adhesion and growth signals, modulating its protein interactions and downstream signaling. It plays critical roles in cytoskeletal organization, cell spreading, and directional migration. Aberrant paxillin expression and phosphorylation are associated with cancer progression, invasion, and metastasis across multiple tumor types. Targeting paxillin-mediated signaling pathways represents a potential strategy for inhibiting cancer cell motility and metastatic dissemination. The AlphaLISA SureFire Ultra Human and Mouse Phospho-Paxillin (Tyr118) is a sandwich immunoassay for the quantitative detection of phospho-Paxillin (Tyr118) 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 Phospho-AlphaLISA SureFire Ultra assay principle The Phospho-AlphaLISA SureFire Ultra assay measures a protein target when phosphorylated at a specific residue. The assay uses two antibodies which recognize the phospho epitope and a distal epitope 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 phosphorylated 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 phosphoprotein present in the sample. Phospho-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 Phospho-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. Phospho-AlphaLISA SureFire Ultra one-plate assay protocol Detection of Phosphorylated 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 Basal Phospho (Tyr118) Paxillin levels 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 lysed with 200 µL of Lysis Buffer. Suspension cells were seeded at 400,000 cells/well in a 96-well culture plate in HBSS + 0.1% BSA, cells were spun down and lysed with 200 µL of Lysis Buffer. Paxillin Total levels were evaluated by AlphaLISA SureFire Ultra . For the detection step, 10 µL of cell lysate (approximately 2,000 adherent cells or 20,000 suspension cells) was 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, high basal Phospho (Tyr118) Paxillin levels was observed in A431 and C2C12 cells while low levels were detected in RT4 and HUVEC cells.
