AlphaLISA SureFire Ultra Human Total DDR1 Detection Kit, 50,000 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 enables cell 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 robust AlphaLISA SureFire Ultra quality.

T47D cells were seeded in a 96-well plate (80,000 cells/well) in complete medium and incubated overnight at 37°C, 5% CO2. The cells were treated with increasing concentrations of Collagen I (derived from rat tail) for 90 minutes.

After treatment, the cells were lysed with 100 µL of Lysis Buffer for 10 minutes at RT with shaking (350 rpm). DDR1 Phospho (Tyr513, Tyr796) 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, Collagen triggered a dose-dependent increase in the levels of Phospho DDR1 (Tyr513, Tyr796) while total levels remained unchanged.

T47D cells were seeded in a 96-well plate (80,000 cells/well) in complete medium and incubated overnight at 37°C, 5% CO2. The cells were treated with increasing concentrations of Pervanadate for 30 minutes.

After treatment, the cells were lysed with 100 µL of Lysis Buffer for 10 minutes at RT with shaking (350 rpm). DDR1 Phospho (Tyr513, Tyr796) 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 DDR1 (Tyr513, Tyr796) while DDR1 Total levels remained unchanged.

Inhibition of DDR1 phosphorylation in Dasatinib treated cells

T47D cells were seeded in a 96-well plate (80,000 cells/well) in complete medium and incubated overnight at 37°C, 5% CO2. The cells were treated for 1 hour with increasing concentrations of Dasatinib followed by treatment with 50 µg/mL Collagen I (derived from rat tail) for 90 minutes.

After treatment, the cells were lysed with 100 µL of Lysis Buffer for 10 minutes at RT with shaking (350 rpm). DDR1 Phospho (Tyr513 and Tyr796) 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, Dasatinib triggered a dose-dependent decrease in the levels of Phospho DDR1 (Tyr513 and Tyr796) while Total DDR1 levels remained unchanged.

Knockout validation of DDR1 Total assay

DDR1 levels were assessed in MCF7 wild type (WT) and MCF7 DDR1 knockout (KO) (Abcam, ab280048) cells. Cells were seeded in a 96-well plate (40,000 cells/well) in complete medium and incubated overnight at 37°C, 5% CO2.

The cells were lysed with 100 µL of Lysis Buffer for 10 minutes at RT with shaking (350 rpm). DDR1 levels were evaluated using the AlphaLISA SureFire Ultra assay. 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 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.

DDR1 was detected in WT but not in the KO cells. This confirms the selectivity of the assay for the detection of DDR1 protein.

Versatility of Total DDR1 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 (200 µL/well) in a 96-well culture plate in HBSS + 0.1% BSA and lysed with 100 µL of Lysis Buffer.

DDR1 levels were evaluated by AlphaLISA SureFire Ultra. For the detection step, 10 µL of cell lysate (approximately 4,000 cells (adherent) or 40,000 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, DDR1 is predominantly expressed in epithelial cell lines.