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AlphaLISA SureFire Ultra Human and Mouse Total AXIN-1 Detection Kit, 500 Assay Points

The AlphaLISA™ SureFire® Ultra™ Human and Mouse Total AXIN-1 assay is a sandwich immunoassay for quantitative detection of total AXIN-1 in cellular lysates using Alpha Technology.

Feature Specification
Application Cell Signaling
Protocol Time 2h at RT

The AlphaLISA™ SureFire® Ultra™ Human and Mouse Total AXIN-1 assay is a sandwich immunoassay for quantitative detection of total AXIN-1 in cellular lysates using Alpha Technology.

Product Variants
Unit Size: 100 assay points
Part #:
ALSU-TAXIN1-A-HV
List Price
USD 694.00
Unit Size: 500 assay points
Part #:
ALSU-TAXIN1-A500
List Price
USD 2,346.00
Unit Size: 10,000 assay points
Part #:
ALSU-TAXIN1-A10K
List Price
USD 14,270.00
Unit Size: 50,000 assay points
Part #:
ALSU-TAXIN1-A50K
List Price
USD 46,060.00
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).
USD 2,346.00
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Overview

Axis inhibition protein 1 (AXIN-1) is a scaffold protein essential for regulating the WNT/β-catenin pathway. It forms the destruction complex with APC, GSK3β, and CK1α to mediate β-catenin degradation, thereby controlling cell proliferation and differentiation. Upon activation of WNT signaling, this complex disassembles, allowing β-catenin to activate the transcription of target genes involved in cell survival and growth. Dysregulation of AXIN-1 can drive tumorigenesis and is associated with neurodegenerative diseases like Alzheimer's due to its role in maintaining cellular homeostasis.

The AlphaLISA SureFire Ultra Human and Mouse Total AXIN-1 Detection Kit is a sandwich immunoassay for the quantitative detection of total AXIN-1 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

Specifications

Application
Cell Signaling
Automation Compatible
Yes
Brand
AlphaLISA SureFire Ultra
Detection Modality
Alpha
Protocol Time
2h at RT
Shipping Conditions
Shipped in Blue Ice
Target
AXIN-1
Target Class
Phosphoproteins
Target Species
Human
Mouse
Technology
Alpha
Therapeutic Area
Neuroscience
Oncology
Unit Size
500 assay points

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.

Assay Principle Total AlphaLISA SureFire Ultra

 

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.

2plates-assay-protocol-AlphaLISA-Surefire-Ultra.jpg
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.

1 plate assay protocol AlphaLISA Surefire Ultra

Assay validation

Increase of AXIN-1 Total levels in XAV-939 treated cells

MCF7 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 a Tankyrase inhibitor (XAV-939) for 24 hours.

After treatment, the cells were lysed with 100 µL of Lysis Buffer for 10 minutes at RT with shaking (350 rpm). AXIN-1 and Cofilin Total levels were evaluated using respective AlphaLISA SureFire Ultra assays. Lysate was further diluted 1:10 in Lysis Buffer for the Cofilin assay. For the detection step, 10 µL of cell lysate (approximately 4,000 cells for AXIN-1 and 400 cells for Cofilin) 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, treatment with XAV-939 triggered a dose-dependent increase in the levels of AXIN-1 while Total Cofilin levels remain unchanged.

Increase of AXIN-1 Total levels in XAV-939 Treated Cells
Specificity of AXIN-1 Total assay

Specificity of the Total AXIN-1 assay was assessed by assaying AXIN-1 and AXIN-2 recombinant human proteins.

Recombinant AXIN-1 (Abcam, ab204090) and AXIN-2 (Abcam, ab204097) proteins were prepared in Lysis Buffer at 500 ng/mL. AXIN-1 Total levels were evaluated using the AlphaLISA SureFire Ultra assay. For the detection step, 10 µL of protein 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.

Total AXIN-1 assay reacts only to AXIN-1 recombinant protein with no cross-reactivity observed to AXIN-2 recombinant protein. These results demonstrate the specificity of the AXIN-1 Total assay as these two proteins share approximately 50% sequence identity.

Selectivity of AXIN-1 Total Assay
Versatility of Total AXIN-1 assay in various cell lines

Various cell lines were seeded at 40,000 cells/well in a 96-well plate in complete medium and incubated overnight at 37°C, 5% CO2. Cells were lysed with 100 µL of Lysis Buffer.

AXIN-1 Total levels were evaluated using the AlphaLISA SureFire Ultra assay. 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.

AXIN-1 is expressed across a wide range of human and mouse cell types. High levels of Total AXIN-1 were detected in SW 48, Hep G2 and MEF cell lines.

Versatility of Total AXIN-1 Assay In Various Cell Lines

Resources

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