MATN2 Antibody

What is MATN2 and why is it important in biological research?

MATN2 (matrilin-2) is a non-collagenous extracellular matrix protein that functions as an oligomeric extracellular adaptor protein in the matrix assembly process. In humans, the canonical protein consists of 956 amino acid residues with a molecular mass of approximately 106.8 kDa and exists in up to four different isoforms . MATN2 plays crucial roles in extracellular matrix formation, cellular adhesion, and tissue development and maintenance, making it a significant target for research in cell biology and tissue engineering . The protein undergoes post-translational modifications, primarily glycosylation, which impacts its functional properties . Its importance in biological research stems from its involvement in multiple physiological processes and its dysregulation in various pathological conditions, including musculoskeletal disorders and certain types of cancer .

What are the key considerations when selecting an anti-MATN2 antibody for research?

When selecting an anti-MATN2 antibody for research, several crucial factors should be considered to ensure experimental success. First, determine the specific experimental application, as different antibodies are optimized for different techniques such as Western blot, ELISA, or immunohistochemistry . Second, consider the species reactivity - many MATN2 antibodies recognize human, mouse and rat proteins, but cross-reactivity varies between products . Third, evaluate whether the antibody targets specific domains or regions of MATN2, as some antibodies are designed to recognize the C-terminal region or specific amino acid sequences (e.g., AA 539-648), which may influence detection of different isoforms . Fourth, determine whether a polyclonal or monoclonal antibody better suits your experimental needs - polyclonals offer broader epitope recognition while monoclonals provide higher specificity . Finally, review available validation data for the antibody in your specific application and tissue/cell type to ensure reliable results.

How do researchers distinguish between the four members of the matrilin family in experimental systems?

Distinguishing between the four members of the matrilin family (MATN1, MATN2, MATN3, and MATN4) in experimental systems requires careful selection of detection methods and controls. Researchers typically employ highly specific antibodies that recognize unique epitopes particular to each matrilin protein . When working with MATN2, it's critical to validate antibody specificity by testing against recombinant proteins of all matrilin family members or using knockout models as negative controls. Differential expression patterns also help distinguish these proteins - MATN2 is widely expressed in many tissues, while MATN1 is primarily found in cartilage, MATN3 in cartilage and growth plates, and MATN4 has a more restricted distribution . At the molecular level, while all matrilins share structural similarities as oligomeric adaptor proteins, MATN2 is uniquely characterized by its 956 amino acid sequence in humans and its specific pattern of von Willebrand factor A domains and EGF-like repeats . RNA expression analysis through qPCR can provide complementary evidence of specific matrilin expression when protein detection methods might cross-react.

What is the relationship between MATN2 and other extracellular matrix components?

MATN2 functions as an oligomeric extracellular adaptor protein that facilitates interactions between various extracellular matrix (ECM) components, creating a complex network essential for tissue integrity and function . As a non-collagenous ECM protein, MATN2 interacts with fibrillar collagens, proteoglycans, and other structural proteins to establish proper matrix architecture and mechanical properties . These interactions are critical during tissue development and maintenance, where MATN2 helps organize the three-dimensional arrangement of matrix molecules. In inflammatory conditions, MATN2 expression is upregulated and can modulate the extracellular environment by inducing proinflammatory gene expression in immune cells such as macrophages, potentially contributing to tissue remodeling and repair processes . Understanding these relationships is essential for researchers investigating ECM dynamics in both normal physiology and pathological conditions, as alterations in these interactions may contribute to the development of fibrosis, impaired wound healing, or other matrix-related disorders .

What are the optimal conditions for Western blot detection of MATN2?

For optimal Western blot detection of MATN2, researchers should consider several key parameters to maximize sensitivity and specificity. Sample preparation is critical - use RIPA buffer supplemented with protease inhibitors for cell lysates, while tissue samples may require additional mechanical disruption followed by homogenization . Since MATN2 is a large protein (106.8 kDa) that undergoes glycosylation, use 8-10% polyacrylamide gels with longer run times to achieve adequate separation . For transfer, semi-dry systems may be sufficient, but wet transfer systems often provide better results for this high molecular weight protein. Blocking should be performed with 5% non-fat dry milk or BSA in TBST for 1-2 hours at room temperature . For primary antibody incubation, dilutions typically range from 1:500 to 1:2000 depending on the specific antibody, with overnight incubation at 4°C yielding the best results . Include positive controls (tissues known to express MATN2 such as skin or peripheral nerve) and negative controls (preferably MATN2 knockout samples if available) . Due to MATN2's post-translational modifications, particularly glycosylation, the observed molecular weight may appear higher than the predicted 106.8 kDa, sometimes appearing as multiple bands representing different glycoforms .

How can researchers optimize immunohistochemical detection of MATN2 in different tissue types?

Optimizing immunohistochemical detection of MATN2 across diverse tissue types requires tailored protocols that account for tissue-specific characteristics. For formalin-fixed paraffin-embedded (FFPE) tissues, antigen retrieval is critical - heat-induced epitope retrieval using citrate buffer (pH 6.0) or EDTA buffer (pH 9.0) for 15-20 minutes typically yields good results . For neural tissues, which may express MATN2 during injury or inflammation, milder antigen retrieval conditions are recommended to preserve tissue morphology . Blocking endogenous peroxidase activity with 3% hydrogen peroxide followed by protein blocking with 5-10% normal serum from the secondary antibody host species is essential to minimize background staining . Primary antibody concentration requires optimization for each tissue type - starting dilutions of 1:100 to 1:500 are recommended with overnight incubation at 4°C . For tissues with high endogenous biotin (liver, kidney), use biotin-free detection systems. As MATN2 is primarily an extracellular matrix protein, expect predominantly extracellular staining patterns, though in some cases (particularly in disease states), intracellular staining may also be observed . Include appropriate positive controls (skin, cartilage) and negative controls (primary antibody omission or ideally MATN2 knockout tissue) with each staining run to validate specificity .

How can researchers effectively measure MATN2 expression levels in different experimental models?

Effective measurement of MATN2 expression levels across experimental models requires a multi-modal approach combining protein and mRNA detection methods. For protein quantification, Western blot analysis using validated anti-MATN2 antibodies provides semi-quantitative data, though researchers should normalize results to appropriate loading controls and include standard curves with recombinant MATN2 protein for more accurate quantification . Enzyme-linked immunosorbent assays (ELISA) offer higher sensitivity and better quantitative precision, especially for secreted MATN2 in conditioned media or biological fluids . At the transcriptional level, quantitative PCR (qPCR) using validated MATN2-specific primers allows sensitive detection of mRNA expression, though researchers must remember that post-transcriptional regulation may cause discrepancies between mRNA and protein levels . For spatial expression patterns, immunohistochemistry or immunofluorescence using anti-MATN2 antibodies provides valuable insights, particularly in complex tissues or disease models where expression may be restricted to specific cell types or regions . When comparing MATN2 expression across different experimental conditions, researchers should implement consistent sample collection and processing protocols, as MATN2 expression can be influenced by factors such as cell density, culture conditions, and inflammatory stimuli . For animal models, consider tissue-specific expression patterns and potential differences in MATN2 regulation between species when interpreting results.

How does MATN2 contribute to neuroinflammatory processes, and how can antibodies help study this role?

MATN2 plays a significant role in neuroinflammatory processes by functioning as an endogenous damage-associated molecular pattern (DAMP) that amplifies inflammatory responses. Research has revealed that MATN2 is upregulated by neurons in response to immune-mediated axonal injury, particularly in models of experimental autoimmune encephalomyelitis (EAE), a mouse model of multiple sclerosis . Upon release, MATN2 directly induces proinflammatory gene expression in macrophages through Toll-like receptor 4 (TLR4) signaling and the MyD88-dependent pathway, promoting the expression of key inflammatory mediators including IL-1β, TNF-α, COX2, and iNOS . MATN2 knockout mice exhibit reduced disease severity, less axonal damage, and decreased proinflammatory gene expression following EAE induction, suggesting a causative role in neuroinflammation .

Anti-MATN2 antibodies are instrumental in studying these processes through multiple approaches. Immunohistochemical staining with anti-MATN2 antibodies allows visualization of MATN2 expression patterns in CNS lesions, revealing its upregulation in damaged neural tissue but absence in normal-appearing white matter, as observed in multiple sclerosis patients . Western blot analysis using these antibodies enables quantification of MATN2 protein levels in different disease stages, while neutralizing antibodies can block MATN2 function in experimental models to assess its contribution to neuroinflammation . When combined with cell-specific markers, MATN2 antibodies in immunofluorescence studies can identify which cell types produce and respond to MATN2 during inflammatory processes, providing crucial insights into the cellular mechanisms underlying neuroinflammatory diseases .

What methodological approaches are most effective for studying MATN2's role in cancer progression?

Studying MATN2's role in cancer progression requires a comprehensive methodological toolkit that spans from molecular analysis to in vivo models. Immunohistochemical analysis of patient-derived tumor samples using validated anti-MATN2 antibodies represents a foundational approach, allowing researchers to correlate MATN2 expression patterns with clinical parameters such as tumor grade, invasiveness, and patient survival . Using multiple antibodies targeting different MATN2 domains can provide insights into potential isoform-specific functions in the tumor microenvironment. For functional studies, researchers should combine gain-of-function approaches (overexpression of MATN2 in cancer cell lines) with loss-of-function strategies (CRISPR/Cas9-mediated knockout or siRNA knockdown) to examine effects on proliferation, migration, invasion, and resistance to apoptosis .

Three-dimensional culture systems, including organoids and spheroids, offer superior models compared to traditional 2D cultures for studying MATN2's influence on tumor architecture and cell-matrix interactions . Co-culture systems incorporating cancer cells with stromal components (fibroblasts, immune cells, endothelial cells) can reveal how MATN2 mediates communication within the tumor microenvironment. In vivo models should include orthotopic xenografts using MATN2-manipulated cancer cells, allowing assessment of tumor growth, metastasis, and the efficacy of targeting MATN2 therapeutically . Throughout these studies, researchers should employ multiple antibodies targeting different MATN2 epitopes and validate findings with complementary approaches such as RNA analysis to ensure robust and reproducible results .

How can researchers distinguish between normal and pathological functions of MATN2 in tissue development and disease?

Distinguishing between normal and pathological functions of MATN2 requires sophisticated experimental approaches that integrate temporal, spatial, and quantitative analyses. Researchers should first establish comprehensive expression maps of MATN2 in normal tissues across developmental stages using a combination of immunohistochemistry, in situ hybridization, and quantitative proteomics . These baseline profiles can then be compared with expression patterns in disease states, noting not only changes in expression levels but also alterations in cellular localization, post-translational modifications, and protein-protein interactions .

Temporal regulation is particularly important—MATN2 often shows dynamic expression during development that differs from its expression in adult tissues or during pathological processes . Conditional and inducible genetic models offer powerful tools for dissecting these temporal aspects, allowing researchers to manipulate MATN2 expression at specific developmental stages or disease timepoints . Using domain-specific antibodies can reveal whether certain functional regions of MATN2 are differentially involved in normal versus pathological processes . Co-localization studies with markers of tissue homeostasis, inflammation, or fibrosis can help contextualize MATN2 expression within broader physiological or pathological processes .

Functional studies comparing MATN2's effects on cellular behavior (adhesion, migration, differentiation) between normal and disease-derived cells can further illuminate context-dependent functions. Importantly, researchers should consider that MATN2's functions may be highly dependent on the composition of the surrounding extracellular matrix, which typically differs between normal and disease states . Integrating these approaches will help distinguish between MATN2's physiological roles in matrix organization and its pathological contributions to diseases like fibrosis, neuroinflammation, and cancer .

What are the key considerations when targeting MATN2 for therapeutic development, and how can antibodies facilitate this research?

When targeting MATN2 for therapeutic development, researchers must address several critical considerations to ensure efficacy and safety. First, understanding the tissue-specific expression patterns of MATN2 is essential since this protein plays diverse physiological roles across multiple tissues . Comprehensive mapping of MATN2 expression in both normal and pathological conditions using well-characterized antibodies helps identify potential off-target effects of therapeutic interventions . Second, the functional redundancy among matrilin family members must be considered—MATN2 inhibition might be compensated by other matrilins, potentially limiting therapeutic efficacy .

Domain-specific antibodies can help determine which functional regions of MATN2 are crucial for pathological processes while minimizing interference with physiological functions . For developing therapeutic antibodies, researchers should establish in vitro screening systems that recapitulate the disease-specific microenvironment, since MATN2's functions are highly context-dependent . Epitope mapping using a panel of antibodies targeting different MATN2 domains can identify the most promising regions for therapeutic targeting . Additionally, researchers must consider the accessibility of MATN2 in different tissues—while it is an extracellular protein theoretically amenable to antibody targeting, the dense architecture of certain matrices may limit antibody penetration .

Pharmacokinetic and pharmacodynamic studies require reliable biomarkers of MATN2 activity, which can be developed using antibody-based assays to monitor therapy response . Finally, since MATN2 participates in both injury response and repair processes, temporal aspects of therapeutic intervention are critical—inhibition during acute inflammatory phases might have different effects than during chronic states, necessitating careful design of preclinical studies with appropriate antibody-based monitoring tools .

What are common challenges in MATN2 antibody-based experiments and how can they be overcome?

Researchers working with MATN2 antibodies frequently encounter several technical challenges that can compromise experimental outcomes. One major issue is non-specific binding, particularly in Western blot applications, which often manifests as multiple bands of unexpected molecular weights . This can be addressed by implementing more stringent blocking conditions (5% BSA instead of milk, which may contain glycoproteins that cross-react), increasing wash duration and frequency, and titrating primary antibody concentrations to optimize signal-to-noise ratios . Another common challenge is inconsistent detection sensitivity between different experimental replicates, which can be mitigated by standardizing protein extraction protocols, particularly for matrix-associated proteins like MATN2 that may require specialized extraction methods using higher detergent concentrations or matrix-digesting enzymes .

For immunohistochemical applications, high background staining often occurs due to MATN2's abundance in extracellular matrix. This can be overcome by using antigen retrieval optimization specific to each tissue type, employing biotin-free detection systems, and including absorption controls where the antibody is pre-incubated with recombinant MATN2 protein . Batch-to-batch variability in antibody performance is another significant challenge, especially with polyclonal antibodies. Researchers should maintain reference samples with known MATN2 expression levels to validate each new antibody lot and consider using pooled antibody preparations when possible . For detecting specific MATN2 isoforms, which can be difficult due to high sequence homology, epitope-specific antibodies targeting unique regions of each isoform should be employed, coupled with appropriate positive controls expressing only the isoform of interest .

How can researchers validate the specificity of anti-MATN2 antibodies in their experimental systems?

Validating anti-MATN2 antibody specificity requires a comprehensive approach combining multiple complementary methods. First, researchers should perform Western blot analysis using tissue or cell lysates with known MATN2 expression alongside negative controls (preferably MATN2 knockout or knockdown samples) . The antibody should detect a band at the expected molecular weight of approximately 106.8 kDa, though post-translational modifications, particularly glycosylation, may cause slight deviations or multiple bands . For polyclonal antibodies, peptide competition assays where the antibody is pre-incubated with the immunizing peptide should eliminate specific signals .

Immunoprecipitation followed by mass spectrometry provides powerful validation by confirming the identity of the protein recognized by the antibody . Cross-reactivity with other matrilin family members (MATN1, MATN3, MATN4) should be assessed using recombinant proteins or cells selectively expressing each family member . For immunohistochemical applications, researchers should compare staining patterns with published literature and confirm concordance between protein detection (using the antibody) and mRNA expression (using in situ hybridization or qPCR on microdissected tissues) .

Different lots of the same antibody should be compared to ensure consistent results, and whenever possible, key findings should be confirmed using multiple antibodies targeting different epitopes of MATN2 . For absolute validation, testing the antibody in MATN2 knockout models or using CRISPR/Cas9-edited cells lacking MATN2 expression provides the most definitive evidence of specificity . Finally, the antibody's performance should be validated specifically for each experimental application (Western blot, immunohistochemistry, ELISA) as specificity can vary between techniques .

What are the best approaches for quantifying MATN2 in complex biological samples?

Quantifying MATN2 in complex biological samples requires tailored approaches based on sample type and research objectives. For absolute quantification in biological fluids or cell culture supernatants, sandwich ELISA using two antibodies recognizing different MATN2 epitopes provides high sensitivity and specificity . Establishing a standard curve with recombinant MATN2 protein allows precise concentration determination, typically in the range of 0.1-10 ng/ml. For tissue samples, Western blot analysis with proper loading controls can provide semi-quantitative data, though researchers should be aware that MATN2's extraction efficiency may vary between tissue types due to differential matrix incorporation .

Advanced mass spectrometry-based approaches, particularly selected reaction monitoring (SRM) or parallel reaction monitoring (PRM), offer superior specificity for quantifying MATN2 in complex proteomes by targeting specific peptide sequences unique to MATN2 . These methods can be particularly valuable when distinguishing between MATN2 isoforms or detecting post-translational modifications. For spatial quantification within tissues, digital image analysis of immunohistochemistry using validated anti-MATN2 antibodies can measure expression levels in specific tissue compartments or cell types .

Multiplex approaches combining MATN2 quantification with other ECM proteins provide valuable context for understanding relative changes in matrix composition. When working with precious or limited samples such as patient biopsies, techniques like proximity extension assay (PEA) or single-molecule array (Simoa) offer ultrasensitive detection capabilities. Regardless of the method chosen, researchers should include appropriate controls (spike-in standards, reference samples) and validate assay performance metrics including linearity, reproducibility, and recovery rates specifically for MATN2 in their sample type of interest .

How do different fixation and sample preparation methods affect MATN2 antibody performance?

For frozen sections, brief fixation (10 minutes in 4% PFA) followed by cryoprotection preserves most MATN2 epitopes while maintaining tissue integrity . When preparing cell lysates for Western blot analysis, extraction buffer composition significantly affects MATN2 recovery - standard RIPA buffers may be insufficient for completely solubilizing matrix-associated MATN2, and stronger detergents (1-2% SDS) or enzymatic treatments (collagenase/hyaluronidase) may be necessary for complete extraction . For immunoprecipitation experiments, milder lysis conditions using NP-40 or Triton X-100 buffers help maintain protein-protein interactions involving MATN2 . Sample storage conditions also impact antibody performance - repeated freeze-thaw cycles can degrade MATN2 or alter its conformation, while long-term storage of fixed tissues may progressively reduce antigenicity . Researchers should therefore optimize and standardize fixation and sample preparation protocols specifically for their MATN2 antibody and experimental system, validating results with appropriate controls.