AlphaLISA SureFire Ultra Human Total TROP2 Detection Kit, 10,000 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.

Selectivity of TROP2 Total assay

TROP2 levels were assessed in MCF7 wild type (WT) and TROP2 KO (Abcam, ab286330) cell lines cultured to confluency in T175 flasks at 37°C, 5% CO₂.

Each flask was lysed in 20 mL of Lysis Buffer for 10 minutes at RT with shaking. Lysates were serially diluted in Lysis Buffer and TROP2 levels were evaluated using the AlphaLISA SureFire Ultra assay kit. For the detection step, 10 µL of cell lysate was transferred into a 384-well white OptiPlate, followed by 5 µL Acceptor Mix and incubated for 1 hour at RT. Finally, 5 µL of Donor Mix was 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.

TROP2 was detected in WT but not in the KO cells. This demonstrates the selectivity of the assay for the detection of TROP2 protein.

Expression of TROP2 in various cell lines

Adherent cells were seeded at 4,000 cells/well in a 96-well culture plate in complete medium and incubated overnight at 37°C, 5% CO₂. Cells were lysed with 200 µL of Lysis Buffer. Suspension cells were washed with HBSS and lysed with Lysis Buffer at 2 x 10⁶ cells/mL.

TROP2 expression was evaluated by AlphaLISA SureFire Ultra. For the detection step, 10 µL of cell lysate (approximately 200 adherent cells or 20,000 suspension 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, TROP2 is highly expressed in A431, RT4 and MCF7 cell lines while no signal was detected in A549, HEK293, THP-1 and RPMI 8226 cells.