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
Phospho-AlphaLISA SureFire Ultra assay principle
The Phospho-AlphaLISA SureFire Ultra assay measures a target protein when phosphorylated at a specific residue in a biological sample (e.g. cell lysate).
The assay uses two antibodies which recognize the phospho epitope and a distal epitope 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 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 for 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 enables cell 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 robust AlphaLISA SureFire Ultra quality.
Induction of Paxillin phosphorylation on fibronectin-coated surface
T25 flasks were coated with 10 µg/mL fibronectin and incubated overnight at 4°C. HeLa cells were seeded in coated or uncoated T25 flasks (1.4 x 106 cells/flask) in serum free medium and incubated for 1 hour at 37°C, 5% CO2.
After incubation, the cells were lysed with 5X Lysis Buffer for 10 minutes at RT with rocking. Paxillin Phospho (Tyr118) and Total levels were evaluated using respective AlphaLISA SureFire Ultra assays. For the detection step, 10 µL of cell lysate (approximately 10,000 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, Phospho (Tyr118) Paxillin was induced in cells seeded onto fibronectin-coated surface, while Total levels remained unchanged.
Phosphorylation of Paxillin in HUVEC cells treated with VEGF
HUVEC cells were seeded in a 96-well plate (20,000 cells/well) in complete medium and incubated overnight at 37°C, 5% CO2. The cells were treated with increasing concentrations of VEGF for 15 minutes.
After treatment, the cells were lysed with 100 µL of Lysis Buffer for 10 minutes at RT with shaking (350 rpm). Paxillin Phospho (Tyr118) and Total levels were evaluated using respective AlphaLISA SureFire Ultra assays. For the detection step, 10 µL of cell lysate (approximately 2,000 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, VEGF triggered a dose-dependent increase in the levels of Phospho (Tyr118) Paxillin while Total levels remained unchanged.
Phosphorylation of Paxillin in cells treated with EGF
HeLa cells were seeded in a 96-well plate (40,000 cells/well) in complete medium and incubated overnight at 37°C, 5% CO2. The cells were starved for 24 hours in serum free media, then treated with increasing concentrations of EGF for 5 minutes.
After treatment, the cells were lysed with 100 µL of Lysis Buffer for 10 minutes at RT with shaking (350 rpm). Paxillin Phospho (Tyr118) and Total levels were evaluated using respective AlphaLISA SureFire Ultra assays. For the detection step, 10 µL of cell lysate (approximately 4,000 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, EGF triggered a dose-dependent increase in the levels of Phospho (Tyr118) Paxillin while Total levels remained unchanged.
Phosphorylation of Paxillin in PANC-1 cells treated with pervanadate
PANC-1 cells were seeded in a 96-well plate (20,000 cells/well) in complete medium and incubated overnight at 37°C, 5% CO2. The cells were treated with increasing concentrations of pervanadate for 15 minutes.
After treatment, the cells were lysed with 100 µL of Lysis Buffer for 10 minutes at RT with shaking (350 rpm). Paxillin Phospho (Tyr118) and Total levels were evaluated using respective AlphaLISA SureFire Ultra assays. For the detection step, 10 µL of cell lysate (approximately 2,000 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, pervanadate triggered a dose-dependent increase in the levels of Phospho (Tyr118) Paxillin while Total levels remained unchanged.
Reduced Paxillin phosphorylation in PANC-1 cells
PANC-1 cells were seeded in a 96-well plate (30,000 cells/well) in complete medium and incubated overnight at 37°C, 5% CO2. The cells were treated with 50 µM Defactinib for 3 hours or 50 µM PF-562271 for 2 hours.
After treatment, the cells were lysed with 100 µL of Lysis Buffer for 10 minutes at RT with shaking (350 rpm). Paxillin Phospho (Tyr118) and Total levels were evaluated using respective AlphaLISA SureFire Ultra assays. For Total Paxillin, lysates were further diluted to work within the assay linear range. For the detection step, 10 µL of cell lysate (approximately 3,000 cells for Phospho Paxillin or 750 cells for Total Paxillin) 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.
Treatment with FAK inhibitors, Defactinib and PF-562271, resulted in a decrease in the levels of Paxillin Phospho (Tyr118) while Total Paxillin levels remained unchanged.
Knockout validation of Paxillin Phospho (Tyr118) assay
Paxillin Phospho (Tyr118) levels were assessed in A431 Wild Type (WT) and Paxillin knockout (KO) cells (Abcam ab261892). Paxillin KO cells and WT cells were seeded at various densities in a 96 well plate in complete medium and incubated overnight at 37°C, 5% CO2.
The cells were lysed with 100 µL of Lysis Buffer for 10 minutes at RT with shaking (350 rpm). Paxillin Phospho (Tyr118) levels were evaluated by AlphaLISA SureFire Ultra. For the detection step, 10 µL of cell lysate 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.
Paxillin Phospho (Tyr118) was only detected in WT cells demonstrating assay selectivity.
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.
