Revvity, ALSU-TTNFA-A500, AlphaLISA Human Total TNFAIP3 Detection Kit, 500 Assay Points

Overview
Tumor necrosis factor alpha-induced protein 3 (TNFAIP3), also known as A20, is a ubiquitin-editing enzyme that negatively regulates NF-κB signaling and inflammation. A20 exhibits both deubiquitinase and E3 ligase activities, allowing it to disassemble K63-linked polyubiquitin chains and add K48-linked chains for proteasomal degradation. This dual function is essential for maintaining immune homeostasis and preventing excessive inflammation. Loss-of-function mutations or reduced expression of A20 are associated with autoimmune diseases, lymphomas, and inflammatory conditions. A20 is a key checkpoint in immune signaling and a potential target for modulating chronic inflammation and enhancing anti-tumor responses.
The AlphaLISA SureFire Ultra Human Total TNFAIP3 Detection Kit is a sandwich immunoassay for the quantitative detection of total TNFAIP3 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

AlphaLISA SureFire Ultra kits can be used for:

Cellular kinase assays
Receptor activation studies
High-throughput screening for preclinical studies
How it works
Total-AlphaLISA SureFire Ultra assay principle

The Total-AlphaLISA SureFire Ultra assay measures the expression level of a protein target in a cell lysate.

The Total-AlphaLISA SureFire Ultra assay uses two antibodies which recognize two different distal epitopes 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 streptavidin to capture one of the detection antibodies, which is biotinylated. In the presence of targeted 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 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 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.
Assay versatility
Expression of TNFAIP3 in various cell lines
Adherent cell lines were seeded in a 96-well plate (40,000 cells/well) and incubated overnight at 37°C, 5% CO2. Cells were lysed with 100 µL of Lysis Buffer for 10 minutes at RT with shaking (350 rpm).
Suspension cell lines were seeded in a 96-well plate (400,000 cells/well) in HBSS + 0.1% BSA and then lysed with 100 µL of Lysis Buffer for 10 minutes at RT with shaking (350 rpm).
TNFAIP3 levels were evaluated by AlphaLISA SureFire Ultra. For the detection step, 10 µL 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. Application-Cell Signaling
Automation Compatible-Yes
Brand-AlphaLISA SureFire Ultra
Detection Modality-Alpha
Product Group-Kit
Protocol Time-2h at RT
Sample Volume-10 µL
Shipping Conditions-Shipped in Blue Ice
Target-TNFAIP3
Target Class-Phosphoproteins
Target Species-Human
Technology-Alpha
Therapeutic Area-Inflammation Oncology
Unit Size-500 assay points