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AlphaLISA SureFire Ultra Mouse Phospho-RIPK1 (Ser321) Detection Kit, 100 Assay Points
AlphaLISA SureFire Ultra Mouse Phospho-RIPK1 (Ser321) Detection Kit, 100 Assay Points
AlphaLISA SureFire Ultra Phospho-Protein
The AlphaLISA™ SureFire® Ultra™ Mouse Phospho-RIPK1 (Ser321) assay is a sandwich immunoassay for quantitative detection of phospho-RIPK1 in cellular lysates using Alpha Technology.
| Feature | Specification |
|---|---|
| Application | Cell Signaling |
| Protocol Time | 2h at RT |
| Sample Volume | 30 µL |
The AlphaLISA™ SureFire® Ultra™ Mouse Phospho-RIPK1 (Ser321) assay is a sandwich immunoassay for quantitative detection of phospho-RIPK1 in cellular lysates using Alpha Technology.
Product variants
Unit Size: 100 assay points
Part #:
ALSU-PRIPK1-C-HV
List price
USD 723.00
Your price:
Unit Size: 500 assay points
Part #:
ALSU-PRIPK1-C500
List price
USD 2,441.00
Your price:
Unit Size: 10,000 assay points
Part #:
ALSU-PRIPK1-C10K
List price
USD 14,847.00
Your price:
Unit Size: 50,000 assay points
Part #:
ALSU-PRIPK1-C50K
List price
USD 47,687.00
Your price:
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).
AlphaLISA SureFire Ultra Mouse Phospho-RIPK1 (Ser321) Detection Kit, 100 Assay Points
AlphaLISA SureFire Ultra Phospho-Protein
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Product information
Overview
Receptor-interacting serine/threonine-protein kinase 1 (RIPK1) is a critical regulator of cell death and inflammation. RIPK1 can mediate cell survival, apoptosis, or necroptosis depending on cellular context and post-translational modifications. In its active form, RIPK1 promotes necroptosis by interacting with RIPK3 and MLKL. Dysregulation of RIPK1 activity is implicated in inflammatory and neurodegenerative diseases, as well as in cancer.
The AlphaLISA SureFire Ultra Mouse Phospho-RIPK1 (Ser321) Detection Kit is a sandwich immunoassay for the quantitative detection of phospho-RIPK1 (Ser321) 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
Phospho-AlphaLISA SureFire Ultra assay principle
The Phospho-AlphaLISA SureFire Ultra assay measures a target protein when phosphorylated at a specific residue in a biological sample (e.g. cell lysate).
The assay uses two antibodies which recognize the phospho epitope and a distal epitope on the target 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 for 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 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 detection reagents. This protocol enables cell viability and confluence to be monitored. In addition, lysates from a single well can be used to measure multiple targets.
Phospho-AlphaLISA SureFire Ultra one-plate assay protocol
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 robust AlphaLISA SureFire Ultra quality.
Assay validation
Activation of Phospho RIPK1 (Ser321) in TNFα 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 treated with increasing concentrations of TNFα for 15 minutes.
After treatment, the cells were lysed with 100 µL of Lysis Buffer for 10 minutes at RT with shaking (350 rpm). RIPK1 Phospho (Ser321) and Total levels were evaluated using respective AlphaLISA SureFire Ultra assays. For the detection step, 10 µL of cell lysate (approximately 4,000 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 using standard AlphaLISA settings.
TNFα treatment in RAW 264.7 cells triggered a dose-dependent increase in the levels of Phospho RIPK (Ser321).
Activation of Phospho RIPK1 (Ser321) 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 treated with increasing concentrations of LPS for 15 minutes.
After treatment, the cells were lysed with 100 µL of Lysis Buffer for 10 minutes at RT with shaking (350 rpm). RIPK1 Phospho (Ser321) and Total levels were evaluated using respective AlphaLISA SureFire Ultra assays. For the detection step, 10 µL of cell lysate (approximately 4,000 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 using standard AlphaLISA settings.
Specifications
| Application |
Cell Signaling
|
|---|---|
| Automation Compatible |
Yes
|
| Brand |
AlphaLISA SureFire Ultra
|
| Cellular or Signaling Pathway |
NFκB signaling
|
| Detection Modality |
Alpha
|
| Molecular Modification |
Phosphorylation
|
| Product Group |
Kit
|
| Protocol Time |
2h at RT
|
| Sample Volume |
30 µL
|
| Shipping Conditions |
Shipped in Blue Ice
|
| Target |
RIPK1
|
| Target Class |
Phosphoproteins
|
| Target Species |
Mouse
|
| Technology |
Alpha
|
| Therapeutic Area |
Inflammation
|
| Unit Size |
100 assay points
|
Video gallery
AlphaLISA SureFire Ultra Mouse Phospho-RIPK1 (Ser321) Detection Kit, 100 Assay Points
Resources
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Guide
AlphaLISA SureFire Ultra: the ultimate guide for successful experiments
The definitive guide for setting up a successful AlphaLISA SureFire Ultra assay
Several biological processes are regulated by...
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