NF-κB p65 (RelA) is a key subunit of the NF-κB transcription factor complex, which regulates genes involved in inflammation, immune responses, apoptosis, and cell proliferation. In its inactive state, p65 is sequestered in the cytoplasm by IκB proteins. Upon activation by pro-inflammatory stimuli such as TNFα, IL-1, or pathogen-associated signals, IκB is phosphorylated and degraded, allowing NF-κB p65 to translocate into the nucleus and drive transcription of target genes. Dysregulation of NF-κB p65 activity contributes to chronic inflammation, autoimmune diseases, and cancer, particularly in driving tumor-promoting inflammation and resistance to apoptosis.
The AlphaLISA SureFire Biotin-Free Human and Mouse Phospho-NF-κB p65 (Ser536) Detection Kit is a sandwich immunoassay for the quantitative detection of phospho-NF-κB p65 (Ser536) 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 Biotin-Free kits are compatible with:
Cell and tissue lysates
Antibody modulators
Biotherapeutic antibodies
AlphaLISA SureFire Biotin-Free kits can be used for:
Cellular kinase assays
Receptor activation studies
High-throughput screening for preclinical studies
Phospho-AlphaLISA SureFire Ultra Biotin Free assay principle
The Phospho-AlphaLISA SureFire Ultra Biotin Free assay measures a protein target when phosphorylated at a specific residue.
The assay uses two antibodies which recognize the phospho epitope and a distal epitope on the targeted 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 a proprietary CaptSure 3 agent to capture one of the detection antibodies, which is labeled with CaptSure 3 tag. In the presence of phosphorylated 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 the generation of a luminescent Alpha signal. The amount of light emission is directly proportional to the quantity of phosphoprotein present in the sample.
Phospho-AlphaLISA SureFire Ultra Biotin Free 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 Biotin Free 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 Biotin Free one-plate assay protocol
Detection of Phosphorylated target protein with AlphaLISA SureFire Ultra Biotin Free 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 Phospho NFκB p65 (Ser536) in IL-1β treated cells
Karpas 299 cells were seeded in a 96-well plate (100,000 cells/well) in complete RPMI 1640 medium and treated with increasing concentrations of IL-1β for 10 minutes.
After treatment, the cells were lysed with the addition of 50 µL of 5X Lysis Buffer for 10 minutes at RT with shaking (350 rpm). NFκB p65 Phospho (Ser536) and Total levels were evaluated using respective AlphaLISA SureFire Biotin Free assays. For the detection step, lysate was further diluted 1:2 with Lysis Buffer and 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.
IL-1β treatment triggered a dose-dependent increase in the levels of Phospho-NFκB p65 (Ser536) while total levels remained unchanged.
Activation of Phospho NFκB p65 (Ser536) in TNFα treated cells
HeLa and 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 starved for 2 hours and then treated with increasing concentrations of TNFα for 5 minutes (HeLa) or 15 minutes (RT4).
After treatment, the cells were lysed with 200 µL of Lysis Buffer for 10 minutes at RT with shaking (350 rpm). NFκB p65 Phospho (Ser536) and Total levels were evaluated using respective AlphaLISA SureFire Biotin Free assays. For the detection step, lysate was further diluted 1:4 with Lysis Buffer and 10 µL of cell lysate (approximately 500 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 (Ser536) while total levels remained unchanged.
Activation of Phospho NFκB p65 (Ser536) in LPS treated cells
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 then 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 (Ser536) and Total levels were evaluated using respective AlphaLISA SureFire Biotin Free 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 (Ser536) while total levels remained unchanged.
