AlphaLISA SureFire Ultra Human and Mouse Phospho-NF-κB p65 (Ser536) 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.

PBMCs were isolated from healthy donors using Ficoll® Plaque Plus. Cells were seeded in a 96-well plate (400,000 cells/well) in complete DMEM and treated with 10 ng/mL IL-1β for 10 minutes; 1 µg/mL LPS or 1 µM PMA for 30 minutes.

After treatment, the cells were spun down and lysed with 100 µL Lysis Buffer for 10 minutes at RT with shaking (350 rpm). NFκB p65 Phospho (p-S536) levels were evaluated using the AlphaLISA SureFire Ultra assay. 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.

A modest increase in phospho levels was observed upon treatment with LPS and PMA.

PBMCs were isolated from healthy donors and cultured for 6 days in complete DMEM containing 20 ng/mL M-CSF to differentiate them into macrophages. Macrophages were seeded in a 96-well plate (40,000 cells/well) in complete DMEM and incubated overnight at 37°C, 5% CO₂. Cells were serum starved for 2 hours and then treated with various agonists for 15 minutes.

After treatment, the cells were lysed with 100 µL Lysis Buffer for 10 minutes at RT with shaking (350 rpm). NFκB p65 Phospho (p-S536) 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.

A modest increase in phospho levels was observed upon treatment with IL-1b and LPS.

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 serum starved for 2 hours and then treated with increasing concentrations of TNFα for 5 minutes.

After treatment, the cells were lysed with 100 µL of Lysis Buffer for 10 minutes at RT with shaking (350 rpm). NFκB p65 Phospho (S536) 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, TNFα triggered a dose-dependent increase in the levels of Phospho NFκB p65 (S536), while Total levels remained unchanged.

RT4 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 serum starved for 2 hours and then treated with increasing concentrations of TNFα for 15 minutes.

After treatment, the cells were washed with HBSS and lysed with 200 µL of Lysis Buffer for 10 minutes at RT with shaking (350 rpm). NFκB p65 Phospho (S536) 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, TNFα triggered a dose-dependent increase in the levels of Phospho NFκB p65 (S536), while Total levels remained unchanged.

Activation of Phospho (Ser536) in IL-1β treated cells

Karpas 299 cells were seeded in a 96-well plate (300,000 cells/well) in HBSS + 0.1% BSA and treated with increasing concentrations of IL-1β for 10 minutes.

After treatment, the cells were lysed with the addition of 50 µL 5X Lysis Buffer for 10 minutes at RT with shaking (350 rpm). NFκB p65 Phospho (S536) and Total levels were evaluated using respective AlphaLISA SureFire Ultra assays. For the detection step, 10 µL of cell lysate (approximately 12,000 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, IL-1β triggered a dose-dependent increase in the levels of Phospho NFκB p65 (S536), while Total levels remained unchanged.

Activation of Phospho (Ser536) in mouse macrophages

RAW 264.7 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 treated with increasing concentrations of LPS for 15 minutes.

After treatment, the cells were lysed with 200 µL of Lysis Buffer for 10 minutes at RT with shaking (350 rpm). NFκB p65 Phospho (S536) 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, LPS triggered a dose-dependent increase in the levels of Phospho NFκB p65 (S536), while Total levels remained unchanged.

Assay sensitivity - cell lysate dilution

Cell lysate was prepared from HeLa cells cultured to confluence in T175 flasks in complete medium. Cells were treated with 50 ng/mL TNFα and 20 ng/mL Calyculin A for 10 minutes and then lysed in 10 mL of Lysis Buffer.

Lysates were serially diluted in Lysis Buffer and assayed for NFκB p65 Phospho (Ser536) and Total levels using respective AlphaLISA SureFire Ultra kits. 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/datapoint is indicated on the graph. The dotted line represents assay background. This assay can detect Phospho NFκB p65 (Ser536) down to 50 cells.