Revvity, ALSU-TTRAF3-A10K, AlphaLISA Human and Mouse Total TRAF3 Detection Kit, 10,000 Assay Points

Overview
TNF receptor-associated factor 3 (TRAF3) is an adaptor protein involved in the regulation of immune responses and apoptosis. TRAF3 is a key mediator in the signaling pathways of various receptors, including TNF receptors, Toll-like receptors, and B-cell receptors. It activates the IKK complex, leading to the activation of NF-κB, which then translocates to the nucleus to promote the expression of target genes. TRAF3 plays a significant role in regulating both innate and adaptive immunity. Dysregulation of TRAF3 can lead to autoimmune diseases and chronic inflammatory conditions. Mutations or deletions of TRAF3 can contribute to the development of cancers, including lymphomas and multiple myeloma.
The AlphaLISA SureFire Ultra Human and Mouse Total TRAF3 Detection Kit is a sandwich immunoassay for the quantitative detection of total TRAF3 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
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
Validation of total TRAF3 assay in various human and mouse cell lines
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. Cells were lysed with 100 µL of Lysis Buffer.
Suspension cells were seeded at 100,000 cells/well (200 µL/well) in a 96-well culture plate in HBSS + 0.1% BSA and lysed with 50 µL 5X Lysis Buffer.
TRAF3 total levels were evaluated using the AlphaLISA SureFire Ultra assay. 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.
Total TRAF3 expression is dependent upon cell type. As expected, high levels of Total TRAF3 were observed in HeLa, A431 and THP-1 cells, while no expression was detected in RAMOS or RPMI 8226 cells. Application-Protein Quantification
Automation Compatible-Yes
Brand-AlphaLISA SureFire Ultra
Detection Modality-Alpha
Host Species-Human Mouse
Molecular Modification-Total
Product Group-Kit
Shipping Conditions-Shipped in Blue Ice
Target-TRAF3
Target Class-Phosphoproteins
Target Species-Human
Technology-Alpha
Therapeutic Area-Inflammation
Unit Size-10,000 assay points