AlphaLISA SureFire Biotin-Free Human and Mouse Androgen Receptor Full-length Specific Detection Kit, 10,000 Assay Points

LNCaP cells were seeded in a 96-well plate (60,000 cells/well) in complete medium and incubated overnight at 37°C, 5% CO₂. Cells were treated with increasing concentrations of ARV-110 PROTAC for 18 hours.

After treatment, the cells were lysed with 100 µL of Lysis Buffer for 10 minutes at RT with shaking (350 rpm). Lysates were further diluted and AR-FL and ERK Total levels were evaluated using respective AlphaLISA SureFire Biotin Free assays*. For the detection step, 10 µL of cell lysate (approximately 1,200 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, ARV-110 induced a dose dependent decrease in AR-FL with no significant changes ERK Total levels.

*AR-FL Specific assay only recognizes Full Length AR protein.

MCF7 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 treated with increasing concentrations of ARV-110 PROTAC for 24 hours.

After treatment, the cells were lysed with 100 µL of Lysis Buffer for 10 minutes at RT with shaking (350 rpm). AR-FL and ERK Total levels were evaluated using respective AlphaLISA SureFire Biotin Free assays. For the detection step, 10 µL of cell lysate (approximately 4,000 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, ARV-110 induced a dose dependent decrease in AR-FL with no significant changes in the levels of ERK Total.

Degradation of AR-FL in 22Rv1 cells treated with AR Degrader-8

22Rv1 cells were seeded in a 96-well plate (60,000 cells/well) in complete medium and incubated overnight at 37°C, 5% CO₂. Cells were treated with increasing concentrations of AR Degrader-8 for 24 hours.

After treatment, the cells were lysed with 100 µL of Lysis Buffer for 10 minutes at RT with shaking (350 rpm). Lysates were further diluted and AR-FL and ERK Total levels were evaluated using AlphaLISA SureFire Biotin Free assays. For the detection step, 10 µL of cell lysate (approximately 1,200 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, AR Degrader-8 induced a dose-dependent decrease in AR-FL with no change in ERK Total levels.

AR-FL expression in various cell lines

Adherent cells were grown to confluency in a T175 flask in complete medium and lysed with Lysis Buffer at a density of 0.5 x 10⁶ cells/mL. Suspension cells were harvested, spun down and lysed with Lysis Buffer at a density of 1.6 x 10⁶ cells/mL.

AR-FL levels were evaluated using the AlphaLISA SureFire Biotin Free assay. For the detection step, 10 µL of cell lysate (5,000 adherent and 16,000 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.

AR-FL expression was detected in various cell lines. Elevated levels were observed in LNCaP cells. No expression was detected in PC3 cells (AR negative cell line).

Assay sensitivity - cell lysate dilution

Cell lysate was prepared from LNCaP cells seeded in T175 flasks and cultured to confluence and lysed in 8 mL of Lysis Buffer for 10 minutes at RT with shaking.

Lysate was serially diluted in Lysis Buffer and AR-FL 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 per datapoint is indicated. The dotted line represents assay background. The AR-FL Specific Total assay can detect AR-FL expression in less than 100 cells.