AXIN-2 is a scaffold protein crucial for the WNT/β-catenin signaling pathway, promoting β-catenin degradation by assembling the destruction complex with APC, GSK3β, and CK1α. Dysregulation of AXIN-2 disrupts β-catenin levels, driving cancers such as colorectal cancer. Loss of AXIN-2 function is also observed in breast cancer and neuroblastoma.
The AlphaLISA SureFire Ultra Human Total AXIN-2 Detection Kit is a sandwich immunoassay for the quantitative detection of total AXIN-2 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
Total-AlphaLISA SureFire Ultra assay principle
The Total-AlphaLISA SureFire Ultra assay measures the expression level of a target protein in a biological sample (e.g. cell lysate).
The Total-AlphaLISA SureFire Ultra assay uses two antibodies which recognize two different distal epitopes 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 target 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 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 enables cell 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 robust AlphaLISA SureFire Ultra quality.
Increase of Total AXIN-2 in SW 48 cells
SW 48 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-2 and ERK1/2 Total levels were evaluated using respective AlphaLISA SureFire Ultra assays. 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, treatment with XAV-939 triggered a dose-dependent increase in the levels of AXIN-2 while Total ERK1/2 levels remain unchanged
Increase of Total AXIN-2 in MCF7 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-2 and Cofilin Total levels were evaluated using respective AlphaLISA SureFire Ultra assays. The lysate was further diluted 1:10 for the Cofilin assay. For the detection step, 10 µL of cell lysate (approximately 4,000 cells for AXIN-2 Total and 400 cells for Cofilin Total) 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-2 while Total Cofilin levels remain unchanged.
Increase of Total AXIN-2 in G007-LK treated cells
SW 48 cells were seeded in a 96-well plate (60,000 cells/well) in complete medium, and incubated overnight at 37°C, 5% CO2. The cells were treated with increasing concentrations of a Tankyrase inhibitor (G007-LK) 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-2 and Cofilin Total levels were evaluated using respective AlphaLISA SureFire Ultra assays. For the detection step, 10 µL of cell lysate (approximately 6,000 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.
As expected, treatment with G007-LK triggered a dose-dependent increase in the levels of AXIN-2 while Total Cofilin levels remain unchanged.
Decrease in Total AXIN-2 levels in MG132 treated cells
SW 48 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 XAV-939 (5 µM) or XAV-939 + MG132 (5 µM) for 6 hours.
After treatment, the cells were lysed with 100 µL of Lysis Buffer for 10 minutes at RT with shaking (350 rpm). AXIN-2 Total levels were evaluated using 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.
As expected, dual treatment with XAV-939 and MG132 induced a decrease in the levels of Total AXIN-2 levels.
Specificity of AXIN-2 Total assay
Specificity of the Total AXIN-2 assay was assessed by assaying AXIN-1 and AXIN-2 recombinant human proteins.
Dilutions of recombinant AXIN-1 (Abcam, ab204090) and AXIN-2 (Abcam, ab204097) were prepared in Lysis Buffer. AXIN-2 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-2 assay reacts only to AXIN-2 recombinant protein with no cross-reactivity observed for AXIN-1 protein. These results demonstrate the specificity of the AXIN-2 Total assay as these two proteins share approximately 50% sequence identity.
Versatility of Total AXIN-2 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-2 Total levels were evaluated using the AlphaLISA SureFire Ultra assay. For the detection step, 10 µL of cell lysate 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.
AXIN-2 Total protein expression is varied depending on cell type. AXIN-2 Total is highly expressed in colorectal cancer cell lines e.g. SW 48 and is not expressed in skin cancer cell line A431.
