AlphaLISA SureFire Ultra Human and Mouse Total NF-κB p65 Detection Kit, 100 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 permits the cells 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 AlphaLISA SureFire Ultra quality.

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.

Validation of Total NFκB p65 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.

Validation of Total NFκB p65 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.

Versatility of Total NFκB p65 assay in various cell lines

Adherent cells were grown to confluency in a T175 flask at 37°C, 5% CO₂ and lysed with Lysis Buffer at a density of 0.5 x 10⁶ cells/mL. Suspension cells were harvested, washed in HBSS and lysed with Lysis Buffer at 1.6 x 10⁶ cells/mL.

Total NFκB p65 levels were evaluated using the AlphaLISA SureFire Ultra assay. For the detection step, 10 µL of cell lysate (5,000 adherent and 16,000 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.

Total NFκB p65 expression was detected in a wide range of human and mouse cell lines.

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 Total NFκB p65 down to 100 cells.