ECM1 Antibody, Biotin conjugated

What is ECM1 and what biological significance does it have?

ECM1 is a secreted glycoprotein involved in multiple biological processes, including extracellular matrix formation, angiogenesis, and immune regulation. It functions as a critical factor in maintaining the latency of deposited L-TGFβ and serves as a gatekeeper for homeostasis in healthy liver tissue . ECM1 physically interacts with connective tissue growth factor (CTGF), specifically binding to the C-terminal cysteine knot domain (CT domain) of CTGF . This interaction inhibits integrin αvβ6-mediated TGFβ activation, reducing fibrotic responses in various tissues . Additionally, ECM1 plays a crucial role in T-follicular helper (TFH) cell differentiation by repressing the IL-2–STAT5–Bcl6 signaling pathway, enhancing germinal center responses and antibody production . Mutations in ECM1 have been detected in skin conditions such as lipoid proteinosis and lichen sclerosis, which are associated with higher levels of autoantibody production .

What types of ECM1 antibodies are available for research purposes?

ECM1 antibodies are available in various formats to accommodate different experimental needs:

While the search results don't specifically mention biotin-conjugated ECM1 antibodies, these would represent an important variant designed to enhance detection sensitivity in various assays through biotin-avidin interactions .

How does biotin conjugation affect ECM1 antibody performance compared to unconjugated versions?

Biotin conjugation provides several methodological advantages over unconjugated antibodies. The biotin-avidin system offers one of the strongest non-covalent interactions in biology, providing exceptional sensitivity for detection. This conjugation allows for signal amplification while maintaining antibody specificity when targeting ECM1.

When using biotin-conjugated ECM1 antibodies, researchers can expect:

• Enhanced sensitivity in detection assays, particularly beneficial when studying tissues with low ECM1 expression

• Greater flexibility in experimental design through multi-step staining protocols

• Compatibility with various detection systems including streptavidin-conjugated fluorophores, enzymes, or quantum dots

• Improved signal-to-noise ratio in applications such as immunohistochemistry and flow cytometry

What are the optimal applications for ECM1 antibodies in research?

ECM1 antibodies can be employed across numerous applications with specific dilution recommendations:

These applications can be further optimized when using biotin-conjugated ECM1 antibodies due to the amplification capabilities of the biotin-streptavidin system, particularly beneficial in tissues where ECM1 expression may be low or in experiments requiring multiplexed detection .

What methodological approach should be used when studying ECM1's interaction with CTGF?

When investigating ECM1-CTGF interactions, researchers should consider a multi-method approach:

• Co-immunoprecipitation (Co-IP): Lysates from cells expressing tagged versions of ECM1 and CTGF can be immunoprecipitated using anti-tag antibodies. Research has demonstrated that FLAG-tagged ECM1 protein can be specifically pulled down from cell lysates containing V5-tagged CTGF, confirming their physical interaction .

• Immunofluorescence co-localization: Double immunofluorescent staining can reveal co-localization of ECM1 and CTGF in pericellular regions of co-transfected cells. Such visualization has been successfully performed in rat hepatic progenitor cells (WB-F344) and primary human bile ductular epithelial cells .

• Solid-phase binding assays: This method utilizes purified proteins to test direct interactions. In previous studies, maltose binding protein (MBP)-fused CTGF or its CT mutants were purified from E. coli and coated on plastic surfaces, demonstrating dose-dependent binding with ECM1 .

• Functional assays: To assess the biological significance of ECM1-CTGF interaction, researchers should measure downstream effects such as TGFβ1 activation using luciferase reporter assays or by quantifying the ratio of active to total TGFβ1 protein in conditioned media .

How can ECM1 antibodies be utilized to investigate liver fibrosis mechanisms?

ECM1 antibodies are instrumental in elucidating the mechanisms of liver fibrosis through several methodological approaches:

• Immunohistochemical analysis: ECM1 antibodies can be used to evaluate ECM1 expression patterns in various liver pathologies including alcohol-associated liver disease (ALD), primary biliary cholangitis (PBC), and primary sclerosing cholangitis (PSC). Research has revealed negative correlations between ECM1 and CTGF expression in these conditions .

• Protein interaction studies: Using ECM1 antibodies in pull-down assays helps identify interaction partners that influence fibrotic processes. The interaction between ECM1 and CTGF has been shown to block integrin αvβ6-mediated TGFβ activation, thereby reducing fibrotic responses in vitro .

• Functional analyses in animal models: In experimental models, ECM1 deficiency has been associated with enhanced susceptibility to 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC)-induced cholestasis, with upregulation of CTGF, αvβ6, alpha-smooth muscle actin, and procollagen type I. Conversely, forced expression of ECM1 attenuated ductular reaction and biliary fibrosis .

• TGFβ signaling assessment: ECM1 antibodies can be employed to investigate how ECM1 influences TGFβ signaling pathways during liver injury. ECM1 has been shown to inhibit alcohol-associated fibrosis and TGFβ-mediated deregulation of hepatocyte nuclear factor 4α, preventing the production of fetal p2 promoter-driven isoforms in experimental models .

What protocols are recommended for using ECM1 antibodies in multiplex immunofluorescence studies?

For multiplex immunofluorescence studies involving ECM1 antibodies, especially biotin-conjugated variants, the following methodological considerations are essential:

• Sequential staining protocol:

  • Begin with antigen retrieval using citrate buffer (pH 6.0) or EDTA buffer (pH 9.0)

  • Block endogenous biotin using avidin-biotin blocking kit

  • Apply primary antibodies sequentially with thorough washing between steps

  • For biotin-conjugated ECM1 antibody, use at dilutions of 1:50-200 depending on the application

  • Detect with streptavidin-conjugated fluorophores with distinct emission spectra

  • Include spectral unmixing steps during image acquisition if fluorophore emission spectra overlap

• Panel design considerations:

  • When studying fibrosis, combine ECM1 with markers such as CTGF, integrin αvβ6, and α-SMA

  • For investigating T cell differentiation, combine with markers including CXCR5, PD-1, ICOS, and Bcl6

  • Include appropriate isotype controls matched to each primary antibody species and class

• Image acquisition and analysis:

  • Use confocal microscopy for high-resolution colocalization studies

  • Implement quantitative image analysis to measure co-expression and spatial relationships

  • Apply consistent thresholding across all experimental groups to ensure reliable quantification

How can researchers validate the specificity of ECM1 antibodies for their experimental systems?

Validating antibody specificity is crucial for obtaining reliable results. For ECM1 antibodies, including biotin-conjugated variants, the following comprehensive validation approach is recommended:

• Positive and negative control tissues/cells:

  • Use tissues known to express ECM1 (e.g., skin, liver) as positive controls

  • Utilize tissues from ECM1 knockout animals or cells with CRISPR-mediated ECM1 deletion as negative controls

• Peptide competition assays:

  • Pre-incubate the ECM1 antibody with excess immunizing peptide (e.g., synthetic peptide derived from human ECM1)

  • Apply this mixture in parallel with the regular antibody application

  • Specific signals should be abolished or significantly reduced in the peptide competition group

• Western blot analysis:

  • Confirm detection of bands at the expected molecular weight (~85 kDa for ECM1)

  • Verify absence of non-specific bands that could confound results

  • Compare against recombinant ECM1 protein standards

• Orthogonal validation:

  • Correlate protein expression results with mRNA expression data

  • Use multiple antibodies targeting different epitopes of ECM1

  • Confirm key findings with alternative methodologies (e.g., mass spectrometry)

What are common challenges when using ECM1 antibodies and how can they be addressed?

Researchers frequently encounter several challenges when working with ECM1 antibodies. The following methodological solutions address these issues:

How should researchers optimize immunoprecipitation protocols when using ECM1 antibodies?

Successful immunoprecipitation (IP) with ECM1 antibodies requires careful optimization:

• Lysis buffer selection:

  • For membrane-associated ECM1: Use NP-40 or Triton X-100 based buffers (0.5-1%)

  • For secreted ECM1: Collect conditioned media and concentrate if necessary

  • Include protease inhibitors (complete cocktail) and phosphatase inhibitors if phosphorylation status is relevant

• Antibody binding conditions:

  • Pre-clear lysates with protein A/G beads to reduce non-specific binding

  • Use 2-5 μg of antibody per 500 μg of total protein

  • Incubate antibody with lysate overnight at 4°C with gentle rotation

• Bead selection and handling:

  • For biotin-conjugated ECM1 antibodies: Use streptavidin-coated beads

  • For unconjugated antibodies: Choose protein A/G beads appropriate for antibody species and isotype

  • Use low-binding tubes to minimize protein loss during handling

• Washing and elution:

  • Perform 4-5 washes with decreasing detergent concentration

  • Elute bound proteins with either low pH buffer, SDS sample buffer, or specific peptide competition

  • For co-IP studies: Use gentler elution conditions to preserve protein-protein interactions

How does ECM1's role in T-follicular helper cell differentiation impact immunological research?

ECM1's influence on T-follicular helper (TFH) cell differentiation has significant implications for immunological research, particularly in vaccine development and autoimmune disease studies:

• Mechanisms of action: ECM1 promotes TFH differentiation by down-regulating STAT5 phosphorylation and up-regulating Bcl6 expression. This occurs downstream of IL-6 and IL-21 signaling, which induce ECM1 expression in TFH cells .

• Experimental evidence: In antigen-immunized mouse models, ECM1 deficiency inhibits TFH cell development and impairs germinal center B-cell reactions and antigen-specific antibody production. Conversely, recombinant ECM1 protein administration enhances TFH differentiation and neutralizing antibody production in mice infected with PR8 influenza virus .

• Research applications:

  • Vaccine adjuvant studies: Investigating ECM1 as a potential adjuvant to enhance antibody responses

  • Autoimmune disease models: Examining ECM1's role in excessive TFH responses in conditions like lupus

  • Infectious disease research: Studying how ECM1 modulation affects protective immunity against pathogens

• Methodological considerations: When studying ECM1's role in TFH differentiation, researchers should:

  • Use flow cytometry to assess TFH markers (CXCR5, PD-1, ICOS, Bcl6)

  • Employ ELISA to measure antigen-specific antibody titers

  • Implement immunohistochemistry to visualize germinal center formation

  • Consider transcriptomic analysis to identify downstream mediators of ECM1 function

What are the emerging applications of ECM1 antibodies in cancer research?

ECM1 has been implicated in various malignancies, making ECM1 antibodies valuable tools in cancer research:

• Diagnostic applications:

  • ECM1 expression has been associated with poor prognosis in several cancers

  • Immunohistochemical studies using ECM1 antibodies can help stratify patients based on expression levels

  • Multiplex staining combining ECM1 with other biomarkers can improve diagnostic accuracy

• Mechanistic investigations:

  • ECM1 has been linked to epithelial-mesenchymal transition (EMT)

  • ECM1 antibodies can help elucidate how ECM1 interacts with the tumor microenvironment

  • Studies suggest ECM1 may influence tumor angiogenesis and metastasis

• Therapeutic implications:

  • Neutralizing ECM1 antibodies could potentially disrupt tumor-promoting functions

  • Antibody-drug conjugates targeting ECM1 might offer selective delivery of cytotoxic agents

  • Combining ECM1 targeting with immunotherapy approaches may enhance anti-tumor responses

• Methodological recommendations:

  • Use tissue microarrays to efficiently screen ECM1 expression across multiple tumor samples

  • Implement digital pathology tools for quantitative analysis of ECM1 staining patterns

  • Consider xenograft models to assess how ECM1 modulation affects tumor growth in vivo

How might ECM1 antibodies contribute to developing therapeutic strategies for fibrotic diseases?

The antifibrotic properties of ECM1 suggest several therapeutic applications where ECM1 antibodies could play crucial roles:

• Target validation: ECM1 antibodies can help validate ECM1 as a therapeutic target by:

  • Confirming its expression patterns in fibrotic tissues from various organs

  • Elucidating its interaction with profibrotic factors like CTGF and TGFβ

  • Identifying patient populations that might benefit from ECM1-targeted therapies

• Monitoring therapeutic responses: During antifibrotic treatment, ECM1 antibodies could:

  • Track changes in ECM1 expression as indicators of treatment efficacy

  • Assess modulation of downstream pathways in serial biopsies

  • Serve as companion diagnostics for novel antifibrotic agents

• Delivery system development: For ectopic ECM1 expression therapy, antibodies can:

  • Help optimize delivery vehicles by tracking biodistribution

  • Confirm target engagement in preclinical models

  • Assess durability of therapeutic ECM1 in tissues

• Biomarker development: ECM1 antibodies might enable:

  • Development of non-invasive assays to detect shed ECM1 in biological fluids

  • Correlation of circulating ECM1 levels with disease progression

  • Identification of patient subsets more likely to respond to specific therapies

Research has demonstrated that ectopic ECM1 expression attenuates ductular reaction and biliary fibrosis in animal models, highlighting the therapeutic potential of enhancing ECM1 activity in chronic liver conditions .

What methodological innovations might enhance the utility of ECM1 antibodies in research?

Several technological advancements could significantly expand the research applications of ECM1 antibodies:

• Single-cell analysis techniques:

  • Integration with mass cytometry (CyTOF) for high-dimensional phenotyping

  • Combining with single-cell RNA sequencing to correlate protein expression with transcriptional profiles

  • Development of proximity ligation assays to study ECM1 interactions at the single-cell level

• Advanced imaging approaches:

  • Super-resolution microscopy to visualize ECM1 distribution at nanoscale resolution

  • Intravital imaging to track ECM1 dynamics in living tissues

  • Correlative light and electron microscopy to link ECM1 localization with ultrastructural features

• Functional antibody derivatives:

  • Development of bi-specific antibodies targeting ECM1 and its interaction partners

  • Creation of antibody fragments with enhanced tissue penetration

  • Engineering of intrabodies to track and manipulate ECM1 in living cells

• Computational tools:

  • Machine learning algorithms to analyze complex ECM1 staining patterns

  • Predictive models of ECM1 function based on structural data

  • Network analysis approaches to position ECM1 within relevant signaling pathways

These methodological innovations could help researchers better understand ECM1's diverse biological functions and potentially translate these insights into novel therapeutic strategies.