AlphaLISA SureFire Ultra Human and Mouse Total BTK 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.

Induction of BTK phosphorylation in Anti-IgM treated cells

Ramos cells were washed, resuspended in HBSS + 0.1% BSA at a density of 6 x 10⁶ cells/mL and seeded in a 96-well plate. Cells were treated with increasing concentrations of anti-IgM for 10 minutes.

After treatment, the cells were lysed with the addition of 50 µL of 5X Lysis Buffer for 10 minutes at RT with shaking (350 rpm). BTK Phospho (Y223, Y551) and Total levels were evaluated using respective AlphaLISA SureFire Ultra assays. For the detection step, 10 µL of cell lysate (approximately 24,000 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, anti-IgM triggered an increase in the levels of Phospho (Y223, Y551) BTK whilst Total levels remained unchanged.

Validation of Total BTK assay in Pervanadate treated cells

U937 cells were washed, resuspended in HBSS + 0.1% BSA at a density of 200,000 cells/well and seeded in a 96-well plate. Cells were treated with increasing concentrations of Pervanadate for 30 minutes.

After treatment, the cells were lysed with the addition of 50 µL of 5X Lysis Buffer for 10 minutes at RT with shaking (350 rpm). BTK Phospho (Y223, Y551) and Total levels were evaluated using respective AlphaLISA SureFire Ultra assays. For the detection step, 10 µL of cell lysate (approximately 8,000 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, Pervanadate triggered a dose-dependent increase in the levels of Phospho (Y223, Y551) BTK whilst Total levels remained unchanged.

Specific degradation of BTK using NX-5948 PROTAC

Ramos cells were seeded in a 96-well plate (100,000 cells/well) in complete medium. The cells were treated for 2 hours with 6.7 µM MG132, followed by increasing concentrations of NX-5948 for 2 hours.

After treatment, the cells were lysed with 100 µL of Lysis Buffer for 10 minutes at RT with shaking (350 rpm). BTK and Cofilin levels were evaluated using respective AlphaLISA SureFire Ultra 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, NX-5948 induced a dose-dependent decrease in BTK levels. The lack of protein degradation in the presence of MG132 confirmed that the degradation was driven by the ubiquitin-proteosome system.

PBMCs were isolated from healthy donors using Ficoll® Plaque Plus and seeded in a 96-well plate (400,000 cells/well) in complete DMEM. Cells were treated with increasing concentrations of NX-5948 for 2.5 hours.

After treatment, the cells were lysed with 100 µL of Lysis Buffer for 10 minutes at RT with shaking (350 rpm). BTK and Cofilin levels were evaluated using respective AlphaLISA SureFire Ultra assays. For the detection step, 10 µL of cell lysate (approximately 40,000 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, NX-5948 triggered a dose-dependent decrease in the levels of BTK.

PBMCs were isolated from healthy donors using Ficoll® Plaque Plus and seeded in a 96-well plate (400,000 cells/well) in complete DMEM. Cells were treated with increasing concentrations of GBD-9 for 16 hours.

After treatment, the cells were lysed with 100 µL of Lysis Buffer for 10 minutes at RT with shaking (350 rpm). BTK and Cofilin levels were evaluated using respective AlphaLISA SureFire Ultra assays. For the detection step, 10 µL of cell lysate (approximately 40,000 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, GBD-9 triggered a dose-dependent decrease in the levels of BTK.

Versatility of Total BTK 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 200 µL of Lysis Buffer.

Suspension cells were seeded at 400,000 cells/well in a 96-well culture plate and lysed with 200 µL of Lysis Buffer.

BTK levels were evaluated by AlphaLISA SureFire Ultra. For the detection step, 10 µL of cell lysate (approximately 2,000 cells (adherent) or 20,000 cells (suspension)) 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, BTK expression is measured only in myeloid and lymphoid cells.