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| Feature | Specification |
|---|---|
| Application | Cell Signaling |
| Sample Volume | 30 µL |
The TEK receptor tyrosine kinase (TIE2), encoded by the TEK gene in humans, is primarily expressed on endothelial cells. Upon binding with angiopoietin-1 (Angpt1), TIE2 induces angiogenesis and supports endothelial cell survival. This activation leads to downstream signaling pathways, including MEK/ERK, IKKb/NFkB, and AKT. Consequently, TIE2 contributes to vascular stability, cellular proliferation, inflammation, and cell survival. Elevated levels of Ang1 and/or Ang2 have been observed in patients with various cancers.
The AlphaLISA SureFire Ultra Human Phospho-TIE2 (Tyr992) Detection Kit is a sandwich immunoassay for the quantitative detection of phospho-TIE2 in cellular lysates, using Alpha technology.
Formats:
AlphaLISA SureFire Ultra kits are compatible with:
Alpha SureFire kits can be used for:
The Phospho-AlphaLISA SureFire Ultra 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 streptavidin to capture one of the detection antibodies, which is biotinylated. 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.
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 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 Phosphorylated 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.
HUVEC cells were seeded in a 96-well culture plate (20,000 cells/well) in complete medium and incubated for 48 hours at 37°C, 5% CO2. The cells were further treated with increasing concentrations of Pervanadate for 30 minutes.
After treatment, the cells were lysed with 50 µL of lysis buffer for 10 minutes at RT with shaking at 350 rpm. TIE2 Phospho (Tyr992) and Total levels were evaluated using respective AlphaLISA SureFire Ultra assays. For the detection step, 10 µL of cell lysate (approximately 8,000 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 Nexus™ using standard AlphaLISA settings.
| Application |
Cell Signaling
|
|---|---|
| Automation Compatible |
Yes
|
| Brand |
AlphaLISA SureFire Ultra
|
| Detection Modality |
Alpha
|
| Host Species |
Human
|
| Lysis Buffer Compatibility |
Lysis Buffer
|
| Molecular Modification |
Phosphorylation
|
| Product Group |
Kit
|
| Sample Volume |
30 µL
|
| Shipping Conditions |
Shipped in Blue Ice
|
| Target |
TIE2
|
| Target Class |
Phosphoproteins
|
| Target Species |
Human
|
| Technology |
Alpha
|
| Therapeutic Area |
Oncology
|
| Unit Size |
100 assay points
|
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The definitive guide for setting up a successful AlphaLISA SureFire Ultra assay
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