Discoidin Domain Receptor 1 (DDR1) is a receptor tyrosine kinase activated by collagen in the extracellular matrix. DDR1 modulates cell adhesion, migration, and matrix remodeling, playing a key role in development, tissue repair, and fibrosis. Its activation leads to downstream signaling through MAPK, PI3K/AKT, and NF-κB pathways. Overexpression and mutation of DDR1 have been implicated in various cancers, including breast, lung, and pancreatic cancer, where it contributes to tumor invasion, metastasis, and resistance to therapy. DDR1 is also associated with fibrotic diseases and has emerged as a promising target for anti-fibrotic and anti-cancer therapies.
The AlphaLISA SureFire Ultra Human Total DDR1 Detection Kit is a sandwich immunoassay for the quantitative detection of total DDR1 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
Total-AlphaLISA SureFire Ultra assay principle
The Total-AlphaLISA SureFire Ultra assay measures the expression level of a target protein in a biological sample (e.g. cell lysate).
The Total-AlphaLISA SureFire Ultra assay uses two antibodies which recognize two different distal epitopes 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 target 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 protein present in the sample.
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.
Phosphorylation of DDR1 in endogenous cell systems
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.
