Phospho-DDR1 (Tyr513) Antibody [F16D7]

Application: Reactivity: Human

Lane 1: T47-D, Lane 2: T47-D (Collagen, 20 μg/ml, 18 h)

Experiment Essentials

WB
Recommended SDS-PAGE separating gel concentration: 5%.

Usage Information

Dilution
WB1:1000

Application
WB

Reactivity
Human

Source
Rabbit Monoclonal Antibody

Storage Buffer
PBS, pH 7.2+50% Glycerol+0.05% BSA+0.01% NaN3

Storage (from the date of receipt)
-20°C (avoid freeze-thaw cycles), 2 years

Predicted MW
125 kDa

Positive Control	T47-D cells (collagen, 20 μg/ml, 18 h)
Negative Control	T47-D cells

Experimental Methods

WB
Experimental Protocol: Sample preparation 1. Tissue: Lyse the tissue sample by adding an appropriate volume of ice-cold RIPA/NP-40 Lysis Buffer (containing Protease Inhibitor Cocktail, Phosphatase Inhibitor Cocktail)，and homogenize the tissue at a low temperature or lyse it by sonication on ice, then incubate on ice for 30 minutes. 2. Adherent cell: Aspirate the culture medium and wash the cells with ice-cold PBS twice. Lyse the cells by adding an appropriate volume of RIPA/NP-40 Lysis Buffer (containing Protease Inhibitor Cocktail, Phosphatase Inhibitor Cocktail) , sonicate to lyse the cells, and incubate on ice for 30 minutes. 3. Suspension cell: Transfer the culture medium to a pre-cooled centrifuge tube. Centrifuge and aspirate the supernatant. Wash the cells with ice-cold PBS twice. Lyse the cells by adding an appropriate volume of RIPA/NP-40 Lysis Buffer (containing Protease Inhibitor Cocktail, Phosphatase Inhibitor Cocktail) , sonicate to lyse the cells, and incubate on ice for 30 minutes. 4. Place the lysate into a pre-cooled microcentrifuge tube. Centrifuge at 4°C for 15 min. Collect the supernatant; 5. Remove a small volume of lysate to determine the protein concentration; 6. Combine the lysate with protein loading buffer. Boil 20 µL sample under 95-100°C for 5 min. Centrifuge for 5 min after cool down on ice. Electrophoretic separation 1. According to the concentration of extracted protein, load appropriate amount of protein sample and marker onto SDS-PAGE gels for electrophoresis. Recommended separating gel (lower gel) concentration: 5%. Reference Table for Selecting SDS-PAGE Separation Gel Concentrations 2. Power up 80V for 30 minutes. Then the power supply is adjusted (110 V~150 V), the Marker is observed, and the electrophoresis can be stopped when the indicator band of the predyed protein Marker where the protein is located is properly separated. (Note that the current should not be too large when electrophoresis, too large current (more than 150 mA) will cause the temperature to rise, affecting the result of running glue. If high currents cannot be avoided, an ice bath can be used to cool the bath.) Transfer membrane 1. Take out the converter, soak the clip and consumables in the pre-cooled converter; 2. Activate PVDF membrane with methanol for 1 min and rinse with transfer buffer; 3. Install it in the order of "black edge of clip - sponge - filter paper - filter paper - glue -PVDF membrane - filter paper - filter paper - sponge - white edge of clip"; 4. The protein was electrotransferred to PVDF membrane. ( 0.45 µm PVDF membrane is recommended ) Reference Table for Selecting PVDF Membrane Pore Size Specifications Recommended conditions for wet transfer: 200 mA, 120 min. ( Note that the transfer conditions can be adjusted according to the protein size. For high-molecular-weight proteins, a higher current and longer transfer time are recommended. However, ensure that the transfer tank remains at a low temperature to prevent gel melting.) Block 1. After electrotransfer, wash the film with TBST at room temperature for 5 minutes; 2. Incubate the film in the blocking solution ( recommending 5% BSA solution) for 1 hour at room temperature; 3. Wash the film with TBST for 3 times, 5 minutes each time. Antibody incubation 1. Use primary antibody dilution buffer to prepare the primary antibody working liquid (recommended dilution ratio for primary antibody 1:1000), gently shake and incubate with the film at 4°C overnight; 2. Wash the film with TBST 3 times, 5 minutes each time; 3. Add the secondary antibody to the blocking solution and incubate with the film gently at room temperature for 1 hour; 4. After incubation, wash the film with TBST 3 times for 5 minutes each time. Antibody staining 1. Add the prepared ECL luminescent substrate (or select other color developing substrate according to the second antibody) and mix evenly; 2. Incubate with the film for 1 minute, remove excess substrate (keep the film moist), wrap with plastic film, and expose in the imaging system.

WB

Experimental Protocol:

Sample preparation

1. Tissue: Lyse the tissue sample by adding an appropriate volume of ice-cold RIPA/NP-40 Lysis Buffer (containing Protease Inhibitor Cocktail, Phosphatase Inhibitor Cocktail)，and homogenize the tissue at a low temperature or lyse it by sonication on ice, then incubate on ice for 30 minutes.

2. Adherent cell: Aspirate the culture medium and wash the cells with ice-cold PBS twice. Lyse the cells by adding an appropriate volume of RIPA/NP-40 Lysis Buffer (containing Protease Inhibitor Cocktail, Phosphatase Inhibitor Cocktail) , sonicate to lyse the cells, and incubate on ice for 30 minutes.

3. Suspension cell: Transfer the culture medium to a pre-cooled centrifuge tube. Centrifuge and aspirate the supernatant. Wash the cells with ice-cold PBS twice. Lyse the cells by adding an appropriate volume of RIPA/NP-40 Lysis Buffer (containing Protease Inhibitor Cocktail, Phosphatase Inhibitor Cocktail) , sonicate to lyse the cells, and incubate on ice for 30 minutes.

4. Place the lysate into a pre-cooled microcentrifuge tube. Centrifuge at 4°C for 15 min. Collect the supernatant;

5. Remove a small volume of lysate to determine the protein concentration;

6. Combine the lysate with protein loading buffer. Boil 20 µL sample under 95-100°C for 5 min. Centrifuge for 5 min after cool down on ice.

Electrophoretic separation

1. According to the concentration of extracted protein, load appropriate amount of protein sample and marker onto SDS-PAGE gels for electrophoresis. Recommended separating gel (lower gel) concentration: 5%. Reference Table for Selecting SDS-PAGE Separation Gel Concentrations

2. Power up 80V for 30 minutes. Then the power supply is adjusted (110 V~150 V), the Marker is observed, and the electrophoresis can be stopped when the indicator band of the predyed protein Marker where the protein is located is properly separated. (Note that the current should not be too large when electrophoresis, too large current (more than 150 mA) will cause the temperature to rise, affecting the result of running glue. If high currents cannot be avoided, an ice bath can be used to cool the bath.)

Transfer membrane

1. Take out the converter, soak the clip and consumables in the pre-cooled converter;

2. Activate PVDF membrane with methanol for 1 min and rinse with transfer buffer;

3. Install it in the order of "black edge of clip - sponge - filter paper - filter paper - glue -PVDF membrane - filter paper - filter paper - sponge - white edge of clip";

4. The protein was electrotransferred to PVDF membrane. ( 0.45 µm PVDF membrane is recommended ) Reference Table for Selecting PVDF Membrane Pore Size Specifications

Recommended conditions for wet transfer: 200 mA, 120 min.

( Note that the transfer conditions can be adjusted according to the protein size. For high-molecular-weight proteins, a higher current and longer transfer time are recommended. However, ensure that the transfer tank remains at a low temperature to prevent gel melting.)

Block

1. After electrotransfer, wash the film with TBST at room temperature for 5 minutes;

2. Incubate the film in the blocking solution ( recommending 5% BSA solution) for 1 hour at room temperature;

3. Wash the film with TBST for 3 times, 5 minutes each time.

Antibody incubation

1. Use primary antibody dilution buffer to prepare the primary antibody working liquid (recommended dilution ratio for primary antibody 1:1000), gently shake and incubate with the film at 4°C overnight;

2. Wash the film with TBST 3 times, 5 minutes each time;

3. Add the secondary antibody to the blocking solution and incubate with the film gently at room temperature for 1 hour;

4. After incubation, wash the film with TBST 3 times for 5 minutes each time.

Antibody staining

1. Add the prepared ECL luminescent substrate (or select other color developing substrate according to the second antibody) and mix evenly;

2. Incubate with the film for 1 minute, remove excess substrate (keep the film moist), wrap with plastic film, and expose in the imaging system.

Datasheet & SDS

Biological Description

Specificity
Phospho-DDR1 (Tyr513) Antibody (Rabbit mAb) [F16D7] detects endogenous levels of total DDR1 protein only when it is phosphorylated at Tyr513.

Subcellular Location
Cell membrane, Membrane, Secreted

Uniprot ID
Q08345

Clone
F16D7

Synonym(s)
CAK, CD167, CD167 antigen-like family member A, CD167a, Cell adhesion kinase, DDR, DDR1, HGK2, mammarian carcinoma kinase 10, Mammary carcinoma kinase 10, MCK-10, MCK10, NEP, neuroepithelial tyrosine kinase, PTK3, PTK3A, RTK6, TRK E, TRKE

Background
Phospho-DDR1 (Tyr513) represents the activated state of discoidin domain receptor 1, a non-canonical receptor tyrosine kinase within the DDR family that is distinguished by its unique activation by extracellular matrix collagens rather than soluble growth factors, positioning it as a critical sensor of tissue stiffness and matrix composition in epithelial and stromal cells. The DDR1 protein contains an extracellular discoidin domain that binds diverse collagens including type I, II, III, IV, V, and VI, a single transmembrane helix, and an intracellular kinase domain with an activation loop where Tyr513 resides, serving as a key autophosphorylation site that correlates with kinase activity and downstream signaling propagation. Collagen binding induces DDR1 clustering into dense membrane aggregates, which drives trans-autophosphorylation at Tyr513 and other tyrosine residues independent of classical kinase activation conformations, and this clustering mechanism positions Tyr513 phosphorylation as a direct readout of receptor engagement with fibrillar collagen matrices. Phosphorylation at Tyr513 creates docking sites for SH2 domain-containing proteins, including Src family kinases, Shp-2 phosphatase, and adaptor proteins such as Shc and Grb2, which recruit downstream effectors and initiate PI3K, MAPK, and Akt signaling cascades that control cell adhesion, migration, proliferation, and survival. DDR1-mediated signaling through the Tyr513 phospho-epitope upregulates matrix metalloproteinase expression, drives collagen remodeling, and modulates epithelial-mesenchymal transition, thereby integrating matrix sensing with proteolytic activity and cytoskeletal reorganization during tissue morphogenesis and repair. DDR1 Tyr513 phosphorylation is inhibited by selective small-molecule inhibitors with nanomolar potency, including DDR1-IN-5 and DDR1-IN-6, which suppress autophosphorylation and block downstream oncogenic signaling, establishing this site as a pharmacologically tractable marker of DDR1 activation in cancer and fibrotic disease models. Pathological conditions involving aberrant collagen deposition and matrix remodeling, including breast carcinoma, pulmonary fibrosis, liver fibrosis, and cardiovascular disease, display elevated DDR1 Tyr513 phosphorylation, linking this modification to disease progression through enhanced cell-matrix adhesion, invasive migration, and sustained proliferative signaling in tumor microenvironments and fibrotic tissues. DDR1 expression is restricted to epithelial lineages in normal tissues, particularly in the kidney, lung, gastrointestinal tract, and brain, and its dysregulation leads to altered tissue architecture, impaired branching morphogenesis in mammary gland development, and aberrant immune responses that affect tumor immunity and chronic inflammation.

Background

Phospho-DDR1 (Tyr513) represents the activated state of discoidin domain receptor 1, a non-canonical receptor tyrosine kinase within the DDR family that is distinguished by its unique activation by extracellular matrix collagens rather than soluble growth factors, positioning it as a critical sensor of tissue stiffness and matrix composition in epithelial and stromal cells. The DDR1 protein contains an extracellular discoidin domain that binds diverse collagens including type I, II, III, IV, V, and VI, a single transmembrane helix, and an intracellular kinase domain with an activation loop where Tyr513 resides, serving as a key autophosphorylation site that correlates with kinase activity and downstream signaling propagation. Collagen binding induces DDR1 clustering into dense membrane aggregates, which drives trans-autophosphorylation at Tyr513 and other tyrosine residues independent of classical kinase activation conformations, and this clustering mechanism positions Tyr513 phosphorylation as a direct readout of receptor engagement with fibrillar collagen matrices. Phosphorylation at Tyr513 creates docking sites for SH2 domain-containing proteins, including Src family kinases, Shp-2 phosphatase, and adaptor proteins such as Shc and Grb2, which recruit downstream effectors and initiate PI3K, MAPK, and Akt signaling cascades that control cell adhesion, migration, proliferation, and survival. DDR1-mediated signaling through the Tyr513 phospho-epitope upregulates matrix metalloproteinase expression, drives collagen remodeling, and modulates epithelial-mesenchymal transition, thereby integrating matrix sensing with proteolytic activity and cytoskeletal reorganization during tissue morphogenesis and repair. DDR1 Tyr513 phosphorylation is inhibited by selective small-molecule inhibitors with nanomolar potency, including DDR1-IN-5 and DDR1-IN-6, which suppress autophosphorylation and block downstream oncogenic signaling, establishing this site as a pharmacologically tractable marker of DDR1 activation in cancer and fibrotic disease models. Pathological conditions involving aberrant collagen deposition and matrix remodeling, including breast carcinoma, pulmonary fibrosis, liver fibrosis, and cardiovascular disease, display elevated DDR1 Tyr513 phosphorylation, linking this modification to disease progression through enhanced cell-matrix adhesion, invasive migration, and sustained proliferative signaling in tumor microenvironments and fibrotic tissues. DDR1 expression is restricted to epithelial lineages in normal tissues, particularly in the kidney, lung, gastrointestinal tract, and brain, and its dysregulation leads to altered tissue architecture, impaired branching morphogenesis in mammary gland development, and aberrant immune responses that affect tumor immunity and chronic inflammation.

References
https://pubmed.ncbi.nlm.nih.gov/36454444/ https://pubmed.ncbi.nlm.nih.gov/29467865/

Protein Size (kDa)	PVDF Transfer Membrane Pore Size (μm)
< 10	0.22
10-20	0.22
20-30	0.45
30-45	0.45
45-70	0.45
80-100	0.45
100-140	0.45
> 140	0.45

Tech Support

Handling Instructions

Tel: +1-832-582-8158 Ext:3

If you have any other enquiries, please leave a message.

Protein Size (kDa)	Separating Gel Percentage
4-40	20%
12-45	15%
10-70	12.5%
15-100	10%
25-100	8%
60-210	5%

Phospho-DDR1 (Tyr513) Antibody (Rabbit mAb) [F16D7]