AlphaLISA SureFire Ultra Human Total AXIN-2 Detection Kit, 100 Assay Points

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.

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.

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.