AlphaLISA SureFire Ultra Human and Mouse Total ARID1B 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.

ARID1B expression in various cell lines

Expression of ARID1B was evaluated in various cell lines using the AlphaLISA SureFire Ultra assay kit.

For the detection step, 10 µL of lysate (approximately 5,000 adherent cells and 8,000 suspension cells) 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.

ARID1B was expressed at high levels in all cell lines tested. Similar levels of ARID1B protein were detected in HEK293T (ARID1A-KO) and HEK293T wild type cell lines.

Total ARID1B assay sensitivity - cell lysate dilution

Cell lysate was prepared from HCT-116 cells cultured to confluence in a T175 flasks and lysed in 16 mL of Lysis Buffer for 10 minutes at RT with shaking.

Lysate was serially diluted in Lysis Buffer and ARID1B 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 per datapoint is indicated. The dotted line represents assay background. This assay can detect ARID1B expression in less than 100 cells/datapoint.