AlphaLISA SureFire Ultra Human and Mouse Phospho-Paxillin (Tyr118) Detection Kit, 10,000 Assay Points

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.

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.

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.