AlphaLISA SureFire Biotin-Free Human AR / BRD4 Ternary Complex Detection Kit, 100 Assay Points

AlphaLISA SureFire Ultra Biotin Free complex detection 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 complex AlphaLISA SureFire Ultra Biotin Free detection reagents. This protocol enables cell viability and confluence to be monitored. In addition, lysates from a single well can be used to measure multiple targets.

AlphaLISA SureFire Ultra Biotin Free protein complex detection one-plate assay protocol

Detection of the protein complex with AlphaLISA SureFire Ultra Biotin Free 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 robust AlphaLISA SureFire Ultra Biotin Free assay quality.

Induction of AR/BRD4 Ternary Complex following RIPTAC treatment

LNCaP and 22Rv1 cells were seeded in a 96-well plate (60,000 cells/well) in complete medium and incubated overnight at 37°C, 5% CO₂. The cells were treated with 200 nM of AR/BRD4 RIPTAC for the indicated time points.

After treatment, the cells were lysed with 100 µL of Lysis Buffer for 10 minutes at RT with shaking (350 rpm). AR/BRD4 Ternary Complex levels were evaluated using the AlphaLISA SureFire Biotin Free assay. For the detection step, 10 µL of cell lysate (approximately 12,000 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.

As expected, treatment with AR/BDR4 RIPTAC compound significantly induced AR/BRD4 Ternary Complex formation.

AR/BRD4 Ternary Complex is stable up to 72 hours following treatment

LNCaP cells were seeded in a 96-well plate (60,000 cells/well) in complete medium and incubated overnight at 37°C, 5% CO₂. The cells were treated with 100 nM of AR/BRD4 RIPTAC for 24, 48 and 72 hours.

After treatment, the cells were lysed with 100 µL of Lysis Buffer for 10 minutes at RT with shaking (350 rpm). AR/BRD4 Ternary Complex levels were evaluated using the AlphaLISA SureFire Biotin Free assay. For the detection step, 10 µL of cell lysate (approximately 12,000 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.

The AR/BDR4 Ternary Complex was stable up to 72 hours after treatment. ERK Total levels remained unchanged (data not shown).

Induction of AR/BRD4 Ternary Complex in a dose-dependent manner

LNCaP and 22Rv1 cells were seeded in a 96-well plate (60,000 cells/well) in complete medium and incubated overnight at 37°C, 5% CO₂. The cells were treated with increasing concentrations of AR/BRD4 RIPTAC for 4 hours.

After treatment, the cells were lysed with 100 µL of Lysis Buffer for 10 minutes at RT with shaking (350 rpm). AR/BRD4 Ternary Complex levels were evaluated using the AlphaLISA SureFire Biotin Free assay. For the detection step, 10 µL of cell lysate (approximately 12,000 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.

As expected, treatment with AR/BDR4 RIPTAC compound significantly induced AR/BRD4 Ternary Complex formation while ERK Total levels remained unchanged.

Assay sensitivity - cell lysate dilution

Cell lysate was prepared from LNCaP cells seeded in T175 flasks and cultured to confluence at 37°C, 5% CO₂.Cells were then treated with 100 nM AR/BRD4 RIPTAC for 4 hours and lysed in 4 mL of Lysis Buffer for 10 minutes at RT with shaking.

Lysates were serially diluted in Lysis Buffer and AR/BRD4 Ternary Complex levels were evaluated using the AlphaLISA SureFire Biotin Free assay. For the detection step, 10 µL of lysate 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.

Approximate number of cells/datapoint is indicated on the graph. The assay can detect AR/BRD4 Ternary Complex down to 1,000 cells. Assay background is represented with a dotted line.