The AlphaLISA™ SureFire® Ultra™ Human and Mouse Total TRAF3 assay is a sandwich immunoassay for quantitative detection of total TRAF3 in cellular lysates using Alpha Technology.
For research use only. Not for use in diagnostic procedures. All products to be used in accordance with applicable laws and regulations including without limitation, consumption and disposal requirements under European REACH regulations (EC 1907/2006).
The AlphaLISA™ SureFire® Ultra™ Human and Mouse Total TRAF3 assay is a sandwich immunoassay for quantitative detection of total TRAF3 in cellular lysates using Alpha Technology.
For research use only. Not for use in diagnostic procedures. All products to be used in accordance with applicable laws and regulations including without limitation, consumption and disposal requirements under European REACH regulations (EC 1907/2006).
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:
AlphaLISA SureFire Ultra kits are compatible with:
Alpha SureFire Ultra kits can be used for:
Automation Compatible |
Yes
|
---|---|
Brand |
AlphaLISA SureFire Ultra
|
Detection Modality |
Alpha
|
Host Species |
Human
Mouse
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Shipping Conditions |
Shipped in Blue Ice
|
Target |
TRAF3
|
Therapeutic Area |
Inflammation
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Unit Size |
500 Assay Points
|
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.
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.
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.
HeLa 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 treated with increasing concentrations of TNFa for 18 hours.
After treatment, the cells were lysed with 100 µL of Lysis Buffer for 10 minutes at RT with shaking (350 rpm). TRAF3 total levels were evaluated using 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, TNFa triggered a dose-dependent increase in the levels of Total TRAF3 which led an increase in the levels of Total NFκB2 p100/p52.
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.
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