AlphaLISA SureFire Ultra Human and Mouse Total ARID1A Detection Kit, 500 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 ARID1A Total assay

Total ARID1A levels were assessed in wild type (WT) HEK293T cells and ARID1A knockout (KO) HEK293T cells (Abcam ab266189) cultured to confluency in T175 flasks at 37°C, 5% CO2.

Each flask was lysed with 4 mL of Lysis Buffer B for 10 minutes at RT with shaking. Lysates were then evaluated for ARID1A expression 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 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.

ARID1A was detected only in the HEK293T-WT cells demonstrating assay selectivity.

Versatility of Total ARID1A assay in various cell lines

Adherent 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. Cells were lysed with 100 µL of Lysis Buffer.

Suspension cells were seeded at 400,000 cells/well in a 96-well culture plate in HBSS + 0.1% BSA and lysed with 100 µL of Lysis Buffer.

ARID1A levels were evaluated by AlphaLISA SureFire Ultra. For the detection step, 10 µL of cell lysate (approximately 4,000 adherent cells or 40,000 suspension 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.