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.

Activation of Total NFκB2 p100/p52 in TNFα treated cells

HeLa cells were seeded in a 96-well culture-treated plate (40,000 cells/well) in complete medium, and incubated overnight at 37°C, 5% CO2. The cells were treated with increasing concentrations of TNFα for 18 hours.

After treatment, the cells were lysed with 100 µL of Lysis Buffer for 10 minutes at RT with shaking (350 rpm). NFκB2 p100/p52 Total and NFκB p65 Total levels were evaluated using respective AlphaLISA SureFire Ultra assays. For the detection step, 10 µL of cell lysate (approximately 4,000 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.

As expected, TNFα triggered a dose-dependent increase in the levels of Total NFκB2 p100/p52 while Total NFκB p65 levels remained unchanged.

Validation of Total NFκB2 p100/p52 in LIGHT treated cells

RT4 cells were seeded in a 96-well culture-treated plate (40,000 cells/well) in complete medium, and incubated overnight at 37°C, 5% CO2. The cells were treated with increasing concentrations of LIGHT for 24 hours.

After treatment, the cells were lysed with 100 µL of Lysis Buffer for 10 minutes at RT with shaking (350 rpm). NFκB2 p100 Total, NFκB2 p100/p52 Total, and TRAF3 Total levels were evaluated using respective AlphaLISA SureFire Ultra assays. For the detection step, 10 µL of cell lysate (approximately 4,000 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.

As expected, LIGHT, a NFκB non-canonical pathway activator, triggered a dose-dependent decrease in the levels of Total TRAF3 which led to a decrease in Total NFκB2 p100 and Total NFκB2 p100/p52.

Validation of Total NFκB2 p100/p52 in LIGHT treated cells

The ratio of Total NFκB2 p100 to Total NFκB2 p100/p52 following treatment with 400 ng/mL of LIGHT for 24 hours is indicated.

As expected, LIGHT increased processing of Total p100 into Total p52, leading to a decrease in the levels of Total p100, which is demonstrated by the lower ratio observed in LIGHT treated RT4 cells.

Specificity of Total NFκB2 p100/p52 assay

Total NFκB2 p100/p52 protein levels were assessed in HCT 116 wild type (WT) and HCT 116 NFκB2 p100 knockout (KO) (Abcam ab266883) cells. Both WT and KO cells were seeded in a 96-well plate (40,000 cells/well) and incubated overnight at 37°C, 5% CO2.

After treatment, the cells were lysed with 100 µL of Lysis Buffer for 10 minutes at RT with shaking (350 rpm). NFκB2 p100/p52 Total levels were then evaluated by AlphaLISA SureFire Ultra. 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, Total NFκB2 p100/p52 was only detected in the WT HCT 116 cells but not in the HCT 116 NFκB2 p100 KO cell line, demonstrating assay specificity.

Differential expression of NFκB2 p100/p52 Total assay in various cell lines

Various human and mouse 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. The cells were lysed the following day with 100 µL of Lysis Buffer.

NFκB2 p100/p52 Total levels were evaluated using the AlphaLISA SureFire Ultra assay. For the detection step, 10 µL (approximately 4,000 cells) of cell lysate 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 NFκB2 p100/p52 expression was detected across all human and mouse adherent cell lines tested. A very high level of expression was detected in SW 1573 and NIH/3T3 cells, with lower levels of expression across HEK293T, A431, HeLa, RAW 264.7, SH-SY5Y and HEK293 cells, demonstrating Total NFκB2 p100/p52 assay versatility.