DDR2 Recombinant Monoclonal Antibody

What is DDR2 and what are its primary functions in cellular processes?

DDR2 is a 130 kDa type I transmembrane glycoprotein belonging to the discoidin-like domain containing subfamily of receptor tyrosine kinases. It functions as a cell surface receptor for fibrillar collagen and regulates multiple cellular processes including cell differentiation, extracellular matrix remodeling, cell migration, and proliferation. The mature human DDR2 consists of a 378 amino acid extracellular domain (ECD) containing the discoidin-like domain, a 22 amino acid transmembrane segment, and a 434 amino acid cytoplasmic domain that includes the kinase domain .

DDR2 plays essential roles in normal bone development by regulating osteoblast differentiation and chondrocyte maturation through signaling pathways involving MAP kinases, ultimately leading to the activation of transcription factor RUNX2. Additionally, DDR2 contributes to tissue remodeling through upregulation of collagenases MMP1, MMP2, and MMP13, facilitating cell migration and potentially tumor cell invasion . It also promotes fibroblast migration and proliferation, contributing to wound healing processes .

How do DDR2 antibodies differ from other receptor tyrosine kinase antibodies?

DDR2 antibodies target a unique receptor tyrosine kinase that specifically recognizes and binds to the triple helical structure of collagen rather than soluble growth factors that typically activate most RTKs. Unlike many RTK antibodies that target domains involved in ligand binding, DDR2 antibodies often target the discoidin domain, which is responsible for collagen recognition and binding .

Another distinguishing feature is that DDR2 exhibits a distinct binding mechanism with different collagen types. While it primarily interacts with collagens I, III, and X through its discoidin-like domain, it can also interact with collagen X through regions outside the discoidin domain, specifically recognizing the non-helical NC1 domain of collagen X, which does not lead to receptor autophosphorylation . This dual interaction mechanism makes DDR2 antibodies particularly valuable for studying differential signaling pathways.

What applications are DDR2 recombinant monoclonal antibodies validated for?

DDR2 recombinant monoclonal antibodies have been validated for multiple research applications across different platforms:

These antibodies have demonstrated reactivity with human samples, with many also cross-reacting with mouse and rat DDR2, making them versatile tools for comparative studies across species .

What is the molecular weight range for detecting DDR2 in Western blotting experiments?

When performing Western blotting experiments to detect DDR2, researchers should expect to observe bands in the 120-140 kDa range . This molecular weight corresponds to the full-length, mature, and post-translationally modified DDR2 protein. The variation in observed molecular weight can be attributed to differences in glycosylation states and other post-translational modifications that may vary between cell types and physiological conditions .

It's important to note that the DDR2 protein contains multiple potential sites for glycosylation, which contributes to the higher apparent molecular weight observed in SDS-PAGE compared to the predicted molecular weight based solely on amino acid sequence. When working with different cell lines or tissue samples, slight variations in the observed molecular weight may be expected due to tissue-specific post-translational modifications .

How does collagen binding induce DDR2 signaling, and how can antibodies be used to modulate this interaction?

Collagen binding to DDR2 induces a complex signaling cascade that begins with receptor dimerization and autophosphorylation. The extracellular domain (ECD) of DDR2 exists as a non-covalent dimer in solution, and this dimerization significantly enhances collagen binding affinity . When collagen binds to the discoidin-like domain of DDR2, it triggers autophosphorylation in the cytoplasmic domain, promoting associations with signaling adaptor proteins such as Shc and Src .

Researchers can use DDR2 antibodies to modulate this interaction through several approaches:

• Blocking antibodies: Antibodies targeting the discoidin domain can inhibit collagen binding, preventing downstream signaling

• Conformation-specific antibodies: These can stabilize either active or inactive conformations of DDR2

• Phospho-specific antibodies: Useful for detecting activated (phosphorylated) forms of DDR2 following collagen stimulation

Within collagen II, the D2 period is required for DDR2 binding, while the D1 period is additionally necessary to trigger DDR2 autophosphorylation . This differential requirement provides opportunities for developing antibodies that selectively inhibit either binding or signaling activation. Notably, DDR2-collagen I interaction inhibits collagen fibrillogenesis and alters collagen fiber morphology, suggesting antibodies targeting this interaction could be valuable tools for studying extracellular matrix remodeling .

What are the critical considerations for validating DDR2 antibody specificity in different experimental contexts?

Validating DDR2 antibody specificity is crucial for ensuring reliable experimental results. Several critical considerations include:

A comprehensive validation strategy should incorporate multiple approaches to provide confidence in antibody specificity across different experimental contexts.

How do DDR2 mutations impact antibody selection for cancer research applications?

DDR2 mutations have been identified in several cancer types, including lung squamous cell carcinoma, and can significantly impact antibody selection for cancer research applications. When studying DDR2 in cancer contexts, researchers should consider:

• Epitope location relative to mutation sites: If antibodies target regions containing known cancer-associated mutations, binding efficiency may be compromised. For instance, mutations in the discoidin domain may affect antibodies targeting this region .

• Expression level variations: DDR2 is upregulated in several pathological conditions, including hepatic fibrosis, rheumatoid and osteoarthritis, and smooth muscle cell hyperplasia . Cancer samples may show variable expression levels requiring antibodies with appropriate sensitivity.

• Phosphorylation-state specific detection: Cancer-associated DDR2 mutations may alter phosphorylation patterns. Phospho-specific antibodies can help characterize the activation status of mutant DDR2 variants.

• Truncated protein detection: Some mutations may lead to truncated forms of DDR2. Antibodies targeting different domains should be selected based on the expected protein product.

When studying DDR2 in lung cancer tissues, immunohistochemistry using specific monoclonal antibodies has demonstrated cytoplasmic localization of DDR2, as shown in studies using Mouse Anti-Human DDR2 Monoclonal Antibody at 15 μg/mL . This cytoplasmic localization pattern may differ from the typical membrane localization, highlighting the importance of considering subcellular localization when selecting antibodies for cancer research.

What are the optimal storage and handling conditions for maximizing DDR2 antibody performance and shelf-life?

Proper storage and handling of DDR2 antibodies are critical for maintaining their performance and extending shelf-life. Based on manufacturer recommendations, the following guidelines should be followed:

• Long-term storage: Store antibodies at -20°C to -70°C for optimal long-term stability. DDR2 antibodies can typically be stored for up to 12 months from date of receipt at these temperatures .

• Short-term storage: For short-term use (up to 1 month), store at 2-8°C under sterile conditions after reconstitution .

• Freeze-thaw cycles: Use a manual defrost freezer and avoid repeated freeze-thaw cycles as these can denature the antibody and reduce its activity. Do not aliquot certain antibodies (e.g., DDR2 (E5S1S) Rabbit mAb) .

• Reconstitution: After reconstitution, antibodies can typically be stored for approximately 6 months at -20°C to -70°C under sterile conditions .

• Formulation considerations: Many DDR2 antibodies are supplied in PBS with additives such as glycerol (50%) and sodium azide (0.02%), which help maintain stability. Some are available in carrier-free formulations designed for conjugation applications .

• Working dilutions: Prepare working dilutions on the day of the experiment for optimal results. Do not store diluted antibodies for extended periods.

Following these guidelines will help ensure consistent antibody performance across experiments and maximize the usable lifespan of the reagent.

What optimization strategies should be employed when using DDR2 antibodies for different tissue types and fixation methods?

Optimizing DDR2 antibody protocols for different tissue types and fixation methods requires systematic approach to ensure specific and robust staining:

• Antibody concentration titration:

  • For immunohistochemistry on FFPE human lung tissue, 25 μg/mL Mouse Anti-Human DDR2 Monoclonal Antibody has been reported effective

  • For human lung cancer tissue, 15 μg/mL has been reported effective

  • Always perform a titration series (e.g., 5, 10, 15, 25 μg/mL) to determine optimal concentration for your specific tissue

• Antigen retrieval optimization:

  • Heat-induced epitope retrieval methods may be necessary for formalin-fixed tissues

  • For DDR2 detection, citrate buffer (pH 6.0) or EDTA buffer (pH 9.0) can be tested to determine optimal retrieval conditions

  • Retrieval time should be optimized (typically 10-30 minutes)

• Incubation conditions:

  • For FFPE tissues, overnight incubation at 4°C has been shown effective for DDR2 antibodies

  • For frozen sections, shorter incubation times at room temperature may be sufficient

• Detection system selection:

  • For chromogenic IHC, the Anti-Mouse HRP-DAB Cell & Tissue Staining Kit has been successfully used with DDR2 antibodies

  • Counterstaining with hematoxylin provides good contrast for visualizing tissue architecture

• Tissue-specific considerations:

  • In lung tissue, DDR2 staining has been observed in cells within connective tissue

  • In lung cancer tissue, cytoplasmic staining patterns have been observed

  • Different tissues may require different blocking reagents to minimize background

A systematic optimization approach testing these variables will help establish reliable protocols for specific tissue types and fixation methods.

How can researchers effectively troubleshoot non-specific binding or weak signal issues with DDR2 antibodies?

Troubleshooting DDR2 antibody performance issues requires a systematic approach to identify and address specific problems:

For non-specific binding issues:

• Optimize blocking conditions:

  • Increase blocking duration (1-2 hours at room temperature)

  • Test different blocking reagents (BSA, normal serum, commercial blocking buffers)

  • Consider using species-specific blocking reagents matched to the secondary antibody host

• Adjust antibody concentration:

  • Excessive antibody concentration can lead to non-specific binding

  • Perform titration experiments to determine optimal concentration

  • For Western blotting, 1:1000 dilution has been effective for many DDR2 antibodies

• Increase washing stringency:

  • Add additional washing steps

  • Increase washing buffer volume

  • Consider adding low concentrations of detergent (0.05-0.1% Tween-20)

• Use proper controls:

  • Include isotype controls (e.g., Mouse IgG2B Isotype Control for DDR2 mouse monoclonal antibodies)

  • Include technical negative controls (omitting primary antibody)

For weak signal issues:

• Optimize antigen retrieval:

  • Test different antigen retrieval methods for fixed tissues

  • Extend retrieval time if signal is weak

• Adjust incubation conditions:

  • Extend primary antibody incubation time (overnight at 4°C)

  • Optimize incubation temperature

• Enhance detection sensitivity:

  • Switch to more sensitive detection systems (e.g., polymer-based detection)

  • Consider signal amplification methods (e.g., tyramide signal amplification)

• Check sample preparation:

  • Ensure proper fixation and processing of samples

  • Verify protein denaturation is complete for Western blotting

• Verify target expression:

  • Confirm DDR2 expression in your sample type using literature or database resources

  • Consider using positive control samples with known DDR2 expression

Systematic documentation of troubleshooting steps will help identify the optimal conditions for specific experimental setups.

How should researchers interpret different localization patterns of DDR2 in immunohistochemistry experiments?

Interpretation of DDR2 localization patterns in immunohistochemistry requires careful consideration of cell type, physiological context, and technical factors:

Common DDR2 localization patterns:

• Membrane localization: As a receptor tyrosine kinase, DDR2 is commonly expected to localize to the plasma membrane. Distinct membrane staining may be observed in cells actively engaging with collagen in the extracellular matrix.

• Cytoplasmic localization: DDR2 has been observed with cytoplasmic localization in certain contexts, particularly in lung cancer tissue samples . This may represent:

  • Newly synthesized receptors in the secretory pathway

  • Internalized receptors following ligand binding

  • Altered trafficking in pathological conditions

• Connective tissue-specific expression: In normal lung tissue, DDR2 staining has been specifically localized to cells in connective tissue . This is consistent with DDR2's role in collagen interaction and matrix remodeling.

Interpretive considerations:

• Physiological context: DDR2 expression and localization may change during development, wound healing, or pathological conditions such as fibrosis and cancer.

• Cell type-specific patterns: Different cell types (fibroblasts, chondrocytes, osteoblasts) may show distinct DDR2 localization patterns reflecting their specialized functions.

• Activation state: Activated DDR2 (following collagen binding) may show altered localization compared to inactive DDR2.

• Technical considerations: Fixation methods and antibody epitope may influence observed localization patterns. Cross-validation with multiple antibodies targeting different epitopes can help confirm genuine localization patterns.

When comparing normal versus pathological tissues, changes in DDR2 localization pattern may provide insights into disease mechanisms, particularly in conditions involving tissue remodeling and altered cell-matrix interactions.

What are the best practices for quantifying DDR2 expression and activation in complex tissue samples?

Quantifying DDR2 expression and activation in complex tissue samples requires robust methodological approaches to ensure accurate and reproducible results:

For expression quantification:

• Digital image analysis:

  • Use specialized software (ImageJ, QuPath, HALO) for objective quantification

  • Set consistent thresholds across sample sets

  • Quantify parameters such as staining intensity, percentage of positive cells, and distribution patterns

• Cell type-specific analysis:

  • Consider using multiplex immunohistochemistry or immunofluorescence to co-stain for DDR2 and cell-type markers

  • This allows quantification of DDR2 expression within specific cell populations

• Tissue compartment analysis:

  • Analyze DDR2 expression separately in different tissue compartments (e.g., epithelium vs. stroma)

  • This is particularly important given DDR2's role in connective tissue and cells in the tumor microenvironment

• Controls and normalization:

  • Include appropriate positive and negative controls in each batch

  • Consider including calibration standards for inter-batch normalization

For activation assessment:

• Phospho-specific detection:

  • Use phospho-specific antibodies to detect activated DDR2

  • Quantify the ratio of phosphorylated to total DDR2 as a measure of activation

• Downstream signaling markers:

  • Assess activation of known downstream targets (e.g., upregulation of MMP-1, MMP-2, and MMP-13)

  • Use of serial sections or multiplex staining to correlate DDR2 with these markers

• Functional readouts:

  • Correlate DDR2 expression with functional outcomes such as collagen reorganization or matrix degradation

  • Use specialized assays (e.g., collagen gel contraction, zymography) on tissue-derived cells

• Scoring systems:

  • Develop or adopt standardized scoring systems that account for staining intensity and distribution

  • Consider H-score or Allred scoring approaches, commonly used for receptor quantification

Following these best practices will facilitate meaningful comparisons across different studies and contribute to a more comprehensive understanding of DDR2 biology in complex tissues.

DDR2 antibodies continue to evolve as valuable tools for understanding complex biological processes, with several emerging applications and future directions:

• Single-cell analysis: Integration of DDR2 antibodies into high-dimensional single-cell analyses using mass cytometry or multiplexed imaging will enable deeper understanding of cellular heterogeneity in tissues, particularly in fibrotic diseases and cancer microenvironments where DDR2 plays significant roles.

• Therapeutic development: As DDR2 has been shown to be inhibited by drugs like Gleevec , antibodies that can modulate DDR2 activity may serve as tools for developing targeted therapies, particularly for cancers where DDR2 is implicated in invasion and metastasis.

• Biomarker development: DDR2 expression patterns in diseases such as hepatic fibrosis, rheumatoid and osteoarthritis, and smooth muscle cell hyperplasia suggest potential for DDR2 antibodies in biomarker development and companion diagnostics.

• Mechanical signaling studies: Given DDR2's role in sensing and responding to extracellular matrix properties, antibodies will be increasingly valuable in studying mechanotransduction and how cells respond to matrix stiffness in development and disease.

• Conjugated antibody applications: Carrier-free antibody formats enable development of DDR2-targeted imaging agents, potentially allowing visualization of matrix remodeling processes in real-time or in vivo.

• Structure-function studies: As understanding of DDR2 signaling mechanisms deepens, conformation-specific antibodies may help elucidate the structural changes associated with receptor activation and signaling.