AlphaLISA SureFire Ultra Human Total PAX2 Detection Kit, 10,000 Assay Points

Specificity of PAX2 Total assay

Specificity of the Total PAX2 assay was assessed by assaying recombinant PAX2 and PAX5 proteins. Dilutions of recombinant PAX2 and PAX5 proteins were prepared in Lysis Buffer. PAX2 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 room temperature. 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 PAX2 assay was specific only to PAX2 recombinant protein, no signal was detected with PAX5 recombinant protein. This result demonstrates the specificity of the Total PAX2 SureFire Ultra assay as these two proteins share approximately 70% sequence identity.

Validation of Human PAX2 Total assay in various cell lines

Adherent cell lines were seeded at 40,000 cells/well in a 96-well culture plate in complete medium and incubated overnight at 37°C, 5% CO₂. Cells were lysed with 100 µL of Lysis Buffer.
Suspension cells (Daudi, Ramos) were seeded at 100,000 cells/well (200 µL/well) in a 96-well culture plate in HBSS + 0.1% BSA, cells were spun down and lysed with 100 µL of Lysis Buffer. Suspension cell lysates were further diluted 1:2.5 with Lysis Buffer.

PAX2 Total levels were evaluated by AlphaLISA SureFire Ultra. 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.

Total PAX2 expression is dependent upon cell type. As expected, a very high level of expression was detected in PANC-1 and AN3 CA cell lines, while signal was undetectable in OVCAR-3, Daudi and Ramos cell lines.