AlphaLISA SureFire Ultra Human and Mouse Total p38 MAPK Detection Kit, 500 Assay Points

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 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 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 robust AlphaLISA SureFire Ultra quality.

LPS induced activation of p38 MAPK in mouse macrophages

RAW 264.7 cells were seeded in a 96-well plate (80,000 cells/well) in complete medium and incubated overnight at 37°C, 5% CO₂. Cells were treated with increasing concentrations of LPS for 30 minutes.

After treatment, the cells were lysed with 100 µL of Lysis Buffer for 10 minutes at RT with shaking (350 rpm). p38 MAPK Total and Phospho (Thr180/Tyr182) levels were evaluated using respective AlphaLISA SureFire Ultra assays. For the detection step, 10 µL of cell lysate (approximately 16,000 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, LPS triggered a dose-dependent increase in the levels of Phospho (Thr180/Tyr182) p38 while Total levels remained unchanged.

Decreased levels of Phospho p38 MAPK (Thr180/Tyr182) in Oxozeaenol treated cells

HEK293 cells were seeded in a 96-well plate (20,000 cells/well) in complete medium and incubated for 48 hours at 37°C, 5% CO₂. The cells were treated with increasing concentrations of Oxozeaenol for 2 hours then 0.75 M of sorbitol for 30 minutes.

After treatment, the cells were lysed with 100 µL of Lysis Buffer for 10 minutes at RT with shaking (350 rpm). p38 MAPK Total and Phospho (Thr180/Tyr182) 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, treatment with Oxozeaenol resulted in a dose-dependent decrease in the levels of p38 Phospho (Thr180/Tyr182), while Total p38 MAPK levels remained unchanged.

PBMCs were isolated from healthy donors using Ficoll® Plaque Plus (Merck GE17-1440-02). Cells were seeded in a 96 well plate (400,000 cells/well) and treated with increasing concentrations of SJFα for 24 hours.

After treatment, the cells were spun down and lysed with 100 µL of Lysis Buffer for 10 minutes at RT with shaking (350 rpm). Total p38 MAPK and Cofilin levels were evaluated using respective AlphaLISA SureFire Ultra assays. For the detection step, 10 µL of cell lysate (approximately 40,000 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.

SJFα treatment resulted in a dose-dependent decrease in the levels of p38 MAPK while Cofilin levels remained unchanged.

HeLa 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 SJFα for 24 hours.

After treatment, the cells were lysed with 100 µL of Lysis Buffer for 10 minutes at RT with shaking (350 rpm). Total p38 MAPK and Cofilin levels were evaluated using respective AlphaLISA SureFire Ultra assays. For the detection step, 10 µL of cell lysate (approximately 4,000 cells or 80 cells for Cofilin) 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.

SJFα treatment resulted in a dose-dependent decrease in the levels of p38 MAPK while Cofilin levels remained unchanged.

p38 MAPK expression in various cell lines

Adherent cells were grown to confluence in a T175 flask and lysed with Lysis Buffer at a density of 0.4 x 10⁶ cells/mL. Suspension cells were spun down and lysed with Lysis Buffer at a density of 4 x 10⁶ cells/mL.

p38 MAPK levels were evaluated using AlphaLISA SureFire Ultra. For the detection step, 10 µL of cell lysate (approximately 4,000 adherent cells or 40,000 suspension 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.

p38 MAPK expression was detected in various human and mouse cell lines.

Assay sensitivity - cell lysate dilution

Cell lysate was prepared from HeLa cells cultured to confluence in a T175 flask, treated with 2 µg/mL anisomycin for 15 minutes and lysed in 5 mL of Lysis Buffer for 10 minutes at RT with shaking.

Lysate was serially diluted in Lysis Buffer and p38 MAPK Phospho (Thr180/Tyr182) and 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 p38 MAPK expression in less than 1,000 cells/datapoint.