NF-κ-B2 p100 is a precursor protein of the NF-κ-B family, which plays a key role in regulating immune response, inflammation, and cell survival. p100 can be processed to p52, a transcriptionally active form that enters the nucleus to activate target genes involved in immune response and apoptosis. Dysregulation of NF-κ-B2 is associated with inflammatory diseases, autoimmune diseases, certain lymphomas and multiple myeloma, making it a potential target for therapeutic strategies.
The AlphaLISA SureFire Ultra Human and Mouse Total NFκB p100 Detection Kit is a sandwich immunoassay for the quantitative detection of total NFκB p100 in cellular lysates, using Alpha Technology.
Formats:
The HV (high volume) kit contains reagents to run 100 wells in 96-well format, using a 60 μL reaction volume.
The 500-point kit contains enough reagents to run 500 wells in 384-well format, using a 20 μL reaction volume.
The 10,000-point kit contains enough reagents to run 10,000 wells in 384-well format, using a 20 μL reaction volume.
The 50,000-point kit contains enough reagents to run 50,000 wells in 384-well format, using a 20 μL reaction volume.
AlphaLISA SureFire Ultra kits are compatible with:
Cell and tissue lysates
Antibody modulators
Biotherapeutic antibodies
Alpha SureFire Ultra kits can be used for:
Cellular kinase assays
Receptor activation studies
Screening
Total-AlphaLISA SureFire Ultra assay principle
The Total-AlphaLISA SureFire Ultra assay measures the expression level of a target protein in a biological sample (e.g. cell lysate).
The Total-AlphaLISA SureFire Ultra assay uses two antibodies which recognize two different distal epitopes on the target protein. AlphaLISA assays require two bead types: Acceptor and Donor Beads. Acceptor Beads are coated with a proprietary CaptSure™ agent to specifically immobilize the assay specific antibody, labeled with a CaptSure tag. Donor Beads are coated with streptavidin to capture one of the detection antibodies, which is biotinylated. In the presence of target protein, the two antibodies bring the Donor and Acceptor Beads in close proximity whereby the singlet oxygen transfers energy to excite the Acceptor Bead, allowing for the generation of a luminescent Alpha signal. The amount of light emission is directly proportional to the quantity of protein present in the sample.
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.
Activation of total NFκB2 p100 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 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 while Total NFκB p65 levels remained unchanged.
Validation of total NFκB2 p100 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.
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 p100 into p52, leading to a decrease in the levels of Total NFκB2 p100, which is demonstrated by the lower ratio observed in LIGHT treated RT4 cells.
Specificity of total NFκB2 p100 assay
Total NFκB2 p100 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 Total levels were evaluated using the AlphaLISA SureFire Ultra assay. 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 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 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 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 expression was detected across all human and mouse adherent cell lines tested. A very high level of expression was detected in A431, SW 1573 and NIH/3T3 cells, with slightly lower levels of expression across HEK293T, HeLa, RAW 264.7, SH-SY5Y and HEK293 cells, demonstrating Total NFκB2 p100 assay versatility.
