Discoidin Domain Receptor 2 (DDR2) is a receptor tyrosine kinase activated by fibrillar collagens in the extracellular matrix, particularly collagen types I, II, and III. DDR2 regulates cell adhesion, migration, proliferation, and matrix metalloproteinase (MMP) expression, playing crucial roles in tissue development, wound healing, and fibrosis. Upon collagen binding, DDR2 undergoes sustained autophosphorylation and activates downstream signaling through MAPK, PI3K/AKT, and Src pathways. Mutations and overexpression of DDR2 are implicated in various cancers, including lung squamous cell carcinoma, breast cancer, and head and neck cancers, where it promotes invasion, metastasis, and drug resistance. DDR2 is also associated with fibrotic diseases and skeletal disorders. Emerging therapeutic strategies target DDR2 to inhibit tumor progression and reduce pathological fibrosis.
The AlphaLISA SureFire Ultra Human and Mouse Phospho-DDR2 (Tyr740) is a sandwich immunoassay for the quantitative detection of phospho-DDR2 (Tyr740) in cellular lysates, using Alpha Technology.
Formats:
The HV (high volume) kit contains reagents to run 100 wells in 96-well format, using a 60 μL reaction volume.
The 500-point kit contains enough reagents to run 500 wells in 384-well format, using a 20 μL reaction volume.
The 10,000-point kit contains enough reagents to run 10,000 wells in 384-well format, using a 20 μL reaction volume.
The 50,000-point kit contains enough reagents to run 50,000 wells in 384-well format, using a 20 μL reaction volume.
AlphaLISA SureFire Ultra kits are compatible with:
Cell and tissue lysates
Antibody modulators
Biotherapeutic antibodies
AlphaLISA SureFire Ultra kits can be used for:
Cellular kinase assays
Receptor activation studies
High-throughput screening for preclinical studies
Phospho-AlphaLISA SureFire Ultra assay principle
The Phospho-AlphaLISA SureFire Ultra assay measures a target protein when phosphorylated at a specific residue in a biological sample (e.g. cell lysate).
The assay uses two antibodies which recognize the phospho epitope and a distal epitope on the target protein. AlphaLISA assays require two bead types: Acceptor and Donor Beads. Acceptor Beads are coated with a proprietary CaptSure™ agent to specifically immobilize the assay specific antibody, labeled with a CaptSure tag. Donor Beads are coated with streptavidin to capture one of the detection antibodies, which is biotinylated. In the presence of phosphorylated protein, the two antibodies bring the Donor and Acceptor Beads in close proximity whereby the singlet oxygen transfers energy to excite the Acceptor Bead, allowing for the generation of a luminescent Alpha signal. The amount of light emission is directly proportional to the quantity of phosphoprotein present in the sample.
Phospho-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 Phospho-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.
Phospho-AlphaLISA SureFire Ultra one-plate assay protocol
Detection of Phosphorylated 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.
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 the 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.
As expected, Collagen I triggered a dose-dependent increase in the levels of Phospho DDR2 (Tyr740) with no change to the Total DDR2 levels.
Pervanadate induces Tyr740 phosphorylation in a dose dependent manner
RD and C2C12 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 with increasing concentrations of pervanadate for 30 minutes.
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, pervanadate triggered a dose-dependent increase in the levels of Phospho DDR2 (Tyr740) while total levels remained unchanged.
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 p-Tyr740 and DDR1 p-Tyr796 in relevant cell lines
Various cell lines treated with pervanadate were evaluated for DDR2 Phospho (Tyr740) and DDR1 Phospho (Tyr796) levels using respective AlphaLISA SureFire Ultra assay kits.
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.
