AlphaLISA SureFire Ultra Human Total Nectin-4 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.

Nectin-4 expression in various cell lines

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

Nectin-4 levels were evaluated by AlphaLISA SureFire Ultra. For the detection step, 10 µL of cell lysate (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.

Regulation of Nectin-4 shedding

A431 cells were seeded in a 96-well plate (40,000 cells/well) in complete medium and incubated overnight at 37°C, 5% CO₂. Cells were placed in fresh DMEM and then left untreated or treated with 5 nM PMA -/+ 25 µM GW280264X for 24 hours.

Cell supernatant was harvested and Nectin-4 levels evaluated using AlphaLISA SureFire Ultra. For the detection step, 10 µL of supernatant 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.

As expected, PMA stimulates the shedding of Nectin-4 into the supernatant, and this is inhibited by the metalloprotease inhibitor GW280264X.

Sensitivity of the Total Nectin-4 assay was assessed by evaluating recombinant Nectin-4 protein.

Dilutions of recombinant Nectin-4 protein (Abcam ab307789) were prepared in Lysis Buffer and 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.

Cell lysate was prepared from A431 cells cultured to confluency in a T175 flask and lysed in 4 mL of Lysis Buffer for 10 minutes at RT with shaking.

Lysate was serially diluted in Lysis Buffer and Nectin-4 levels were evaluated by AlphaLISA SureFire Ultra. 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.

Approximate number of cells is indicated. The dotted line represents assay background. The Nectin-4 assay can detect Nectin-4 expression in less than 1,000 cells/datapoint.