AlphaLISA SureFire Ultra Human and Mouse Total DDR2 Detection Kit, 10,000 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 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.

Collagen I induces time dependent phosphorylation of DDR2 (Tyr740)

HT-1080 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 µg/mL of Collagen I (derived from rat tail) for indicated times.

After treatment, the cells were lysed with 50 µL of Lysis Buffer B for 10 minutes at RT with shaking (350 rpm). DDR2 Phospho (Tyr740) and Total levels were evaluated using respective AlphaLISA SureFire Ultra assays. 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, Collagen I triggered a time dependent increase in the levels of Phospho DDR2 (Tyr740) with no change to the Total DDR2 levels.

Collagen I induces DDR2 phosphorylation in a dose dependent manner

HT-1080 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 Collagen I (derived from rat tail) for 4 hours.

After treatment, the cells were lysed with 50 µL of Lysis Buffer B for 10 minutes at RT with shaking (350 rpm). DDR2 Phospho (Tyr740) and Total levels were evaluated using respective AlphaLISA SureFire Ultra assays. 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.

Collagen I treatment triggered a dose-dependent increase in the levels of Phospho DDR2 (Tyr740) with no change to the Total DDR2 levels.

Inhibition of DDR2 phosphorylation in Dasatinib treated cells

HT-1080 cells were seeded in a 96-well plate (50,000 cells/well) in complete medium and incubated overnight at 37°C, 5% CO₂. The cells were treated for 2 hours with increasing concentrations of Dasatinib, all media was removed and then followed by treatment with 100 µg/mL Collagen I (derived from rat tail) for 4 hours.

After treatment, the cells were lysed with 50 µL of Lysis Buffer B for 10 minutes at RT with shaking (350 rpm). DDR2 Phospho (Tyr740) and Total levels were evaluated using respective AlphaLISA SureFire Ultra assays. For the detection step, 10 µL of cell lysate (approximately 10,000 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 DDR2 (Tyr740) while total levels remained unchanged.

Differential expression of DDR2 Total and DDR1 Total in relevant cell lines

Expression of DDR2 and DDR1 was evaluated in various cell lines using the AlphaLISA SureFire Ultra assay kit.

For the detection step, 10 µL of lysate (approximately 50,000 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.

A significant difference in expression profiles was observed between DDR2 and DDR1. As expected, DDR2 was only detected in RD and SH-SY5Y cells while DDR1 expression was observed across various cell lines.