ECM1 Antibody, HRP conjugated

What is ECM1 and what are the primary applications of ECM1 Antibody, HRP conjugated?

ECM1 is a glycoprotein (~75 kDa) involved in many biological processes, particularly in cancer progression and cardiac aging . ECM1 Antibody, HRP conjugated, is primarily used in Western blot analysis, ELISA, and immunohistochemistry.

For Western blot applications, the antibody can detect ECM1 in human tissue lysates as demonstrated with CCD-1070Sk human foreskin fibroblast cell line and WS-1 human fetal skin fibroblast cell line, where a specific band is detected at approximately 75 kDa . These applications typically use reducing conditions and specific immunoblot buffer groups for optimal results.

In ELISA applications, the antibody functions as a capture antibody when paired with complementary detection antibodies. For instance, Mouse Anti-Human ECM-1 Monoclonal Antibody (MAB39371) can be coated on a Clear Polystyrene Microplate to capture recombinant human ECM1 protein for developing standard curves .

What cell lines and tissues show reliable ECM1 expression for positive controls?

Several cancer cell lines and tissues demonstrate consistent ECM1 expression and can serve as reliable positive controls:

For negative controls, normal colonic epithelial cells (NCM460) show lower expression levels compared to colorectal cancer cell lines . Mouse primary cultured cardiac fibroblasts show no ECM1 expression under control conditions, making them suitable negative controls for cardiac research .

What are the optimal sample preparation methods for ECM1 detection?

For optimal ECM1 detection using HRP-conjugated antibodies, sample preparation is crucial:

In Western blot applications:

• Use PVDF membranes for protein transfer

• Apply reducing conditions for optimal epitope exposure

• Probe membranes with 2 μg/mL of Mouse Anti-Human ECM-1 Monoclonal Antibody

• Use appropriate immunoblot buffer groups (e.g., Immunoblot Buffer Group 3)

For cell culture experiments:

• Grow cells to 40-60% confluence before antibody application

• For transfection experiments, verify ECM1 expression at 48 hours post-transfection

For tissue samples:

• Standard formalin-fixed paraffin-embedded (FFPE) protocols are suitable for ECM1 immunohistochemistry

• Consider using antigen retrieval methods to maximize epitope accessibility

How can ECM1 Antibody, HRP conjugated be utilized to study EMT in cancer progression?

EMT (Epithelial-Mesenchymal Transition) is a critical process in cancer metastasis, and ECM1 has been implicated in this mechanism. To study ECM1's role in EMT:

• Design experiments to simultaneously detect ECM1 and EMT markers:

  • E-cadherin (epithelial marker, downregulated during EMT)

  • Vimentin (mesenchymal marker, upregulated during EMT)

• Incorporate analysis of downstream signaling:

  • p-AKT, p-GSK3β, and Snail are downstream targets affected by ECM1 expression

  • Treatment with PI3K inhibitors (LY294002) or activators (740 Y-P) can confirm pathway involvement

Research findings indicate that ECM1 and Vimentin expression are positively correlated in HCC tissues (r = 0.534, P < 0.001), with co-positive expression in 55.0% of cases . When studying EMT, researchers should examine whether ECM1 overexpression leads to decreased E-cadherin and increased Vimentin expression, as demonstrated in both colorectal cancer and hepatocellular carcinoma studies .

How does ECM1 influence cancer cell migration and invasion, and how can this be studied?

ECM1 significantly influences cancer cell migration and invasion through several mechanisms. To investigate these processes:

• Migration assays:

  • Perform wound healing assays after ECM1 overexpression or knockdown

  • Document migration at standardized time points (e.g., 24, 48, 72 hours)

  • Quantify wound closure rate

• Invasion assays:

  • Use Transwell chambers with Matrigel coating

  • Compare invasive potential between ECM1-modified and control cells

  • Analyze matrix metalloproteinase (MMP) expression

• Mechanistic studies:

  • Examine PI3K/AKT/GSK3β/Snail signaling pathway components

  • Use pathway inhibitors/activators to confirm ECM1's role

Research has demonstrated that ECM1 overexpression enhances migration and invasion of hepatocellular carcinoma cells . Similarly, in colorectal cancer, ECM1 knockdown suppressed cell growth, migration, and invasion, while overexpression produced opposite effects .

What approaches are recommended for studying the relationship between ECM1 and signaling pathways?

To investigate ECM1's relationship with signaling pathways:

• Pathway analysis:

  • Use Western blotting with ECM1 Antibody (HRP conjugated) alongside antibodies against key signaling molecules:

    • p-AKT/total AKT

    • p-GSK3β/total GSK3β

    • Snail

• Pathway manipulation:

  • Apply specific inhibitors (e.g., LY294002 for PI3K inhibition)

  • Use activators (e.g., 740 Y-P for PI3K activation)

  • Observe effects on ECM1-mediated cellular changes

• Gene knockdown/overexpression:

  • Implement siRNA against ECM1 using sequences like 5′-CGAGGAGAAGGACTTAAAG-3′ that efficiently reduce ECM1 expression

  • Create overexpression models using lentiviral systems with pLVX-CMV-IRES-EGFP-puro-ECM1 vectors

Evidence suggests that ECM1 regulates colorectal cancer metastasis and EMT processes via the PI3K/AKT/GSK3β/Snail signaling pathway, as treatment with LY294002 and 740 Y-P reversed the effects of ECM1 modulation on CRC cell metastasis .

What are the optimal dilutions and detection systems for ECM1 Antibody, HRP conjugated?

For optimal results when using ECM1 Antibody, HRP conjugated:

As noted in the literature, "Optimal dilutions should be determined by each laboratory for each application" . It's recommended to perform titration experiments to determine the optimal concentration for your specific experimental system.

Why might ECM1 detection show inconsistent results between experiments?

Several factors can contribute to inconsistent ECM1 detection:

• Sample preparation variability:

  • Protein degradation during lysate preparation

  • Incomplete protein denaturation

  • Buffer compatibility issues

• ECM1 isoform differences:

  • Multiple isoforms exist, ranging from ~75 kDa to ~85 kDa

  • Different isoforms may be expressed in different tissues or under various conditions

• Post-translational modifications:

  • ECM1 is a glycoprotein, and glycosylation patterns may vary

  • Phosphorylation states may affect antibody binding

• Technical considerations:

  • Antibody batch variations

  • Suboptimal blocking conditions

  • Secondary antibody cross-reactivity

Research has shown that ECM1 is detected at approximately 75 kDa in fibroblast cell lines , but expression levels can vary significantly between normal and pathological tissues.

What controls are essential when using ECM1 Antibody, HRP conjugated?

Implementing proper controls is crucial for reliable ECM1 detection:

• Positive controls:

  • Cell lines with known ECM1 expression (e.g., CCD-1070Sk, WS-1, SW480, HT29)

  • Recombinant ECM1 protein for standard curves

  • Tissues with verified ECM1 expression (e.g., CRC or HCC tissues)

• Negative controls:

  • NCM460 cells (lower ECM1 expression)

  • Mouse primary cardiac fibroblasts (no ECM1 expression under control conditions)

  • Isotype control antibodies to assess non-specific binding

• Technical controls:

  • Loading controls (β-tubulin ~55kDa is commonly used)

  • Secondary antibody-only controls

  • Blocking peptide controls to confirm specificity

• Experimental controls:

  • ECM1 knockdown samples (using validated siRNA sequences)

  • ECM1 overexpression samples (using lentiviral constructs)

How should variations in ECM1 band size be interpreted in Western blot analysis?

When interpreting ECM1 Western blot results:

• Expected band size:

  • The primary ECM1 band is typically observed at approximately 75 kDa

  • Additional bands may represent:

    • Isoforms (ECM1a, ECM1b, ECM1c)

    • Post-translational modifications

    • Degradation products

• Analytical considerations:

  • Confirm specificity with knockdown experiments

  • Compare with recombinant protein standards

  • Validate across multiple cell lines/tissues

• Glycosylation effects:

  • As a glycoprotein, ECM1 may show varied migration patterns

  • Consider deglycosylation experiments if precise molecular weight is critical

The literature reports consistent detection of ECM1 at approximately 75 kDa in human fibroblast cell lines under reducing conditions .

What statistical approaches are appropriate for analyzing ECM1 expression in patient samples?

For robust statistical analysis of ECM1 expression in patient samples:

• For immunohistochemistry data:

  • Use chi-square test to compare expression between tumor and normal tissues

  • Apply Spearman rank test to assess correlation with other markers (e.g., ECM1 and Vimentin correlation: r = 0.534, P < 0.001)

• For survival analysis:

  • Apply Kaplan-Meier method with log-rank test to compare survival between ECM1-positive and ECM1-negative cases

  • Use Cox proportional hazards model for multivariate analysis

• For clinicopathological correlations:

  • Create contingency tables similar to:

• For quantitative PCR data:

  • Apply Student's t-test for two-group comparisons

  • Use ANOVA with appropriate post-hoc tests for multiple group comparisons

Research has demonstrated that ECM1 expression is significantly associated with TNM stage (P = 0.049) and venous invasion (P = 0.030) in HCC patients, indicating its potential as a prognostic marker .

How can ECM1 expression be quantitatively assessed in relation to EMT markers?

To quantitatively analyze ECM1 in relation to EMT markers:

• Co-expression analysis:

  • Perform multiplexed immunostaining or sequential Western blots

  • Calculate correlation coefficients between ECM1 and EMT markers (E-cadherin, Vimentin)

  • Conduct regression analysis to determine predictive relationships

• Flow cytometry approach:

  • Use t-distributed stochastic neighbor embedding (tSNE) analysis

  • Compare marker expression in ECM1+ versus ECM1- cell populations

  • Generate histograms showing differential expression patterns

• Quantitative scoring methods:

  • Develop H-score or Allred scoring for immunohistochemistry

  • Use densitometry for Western blot quantification

  • Normalize to appropriate housekeeping genes/proteins

Research has identified significant correlations between ECM1 and Vimentin expression in HCC tissues, with co-positive expression in 55.0% of cases (66/120) , providing a foundation for quantitative EMT association studies.

How can ECM1 Antibody, HRP conjugated be used to investigate protein-protein interactions?

To study ECM1 protein interactions:

• Pull-down assays:

  • Use purified ECM1 fragments conjugated to glutathione-sepharose beads

  • Incubate with potential binding partners (e.g., PGRN)

  • Detect interactions through immunoblotting

• Co-immunoprecipitation:

  • Use ECM1 Antibody, HRP conjugated to pull down ECM1 and associated proteins

  • Analyze precipitated proteins by mass spectrometry or Western blot

  • Confirm interactions with reciprocal co-IP experiments

• Proximity ligation assay:

  • Visualize protein-protein interactions in situ

  • Combine ECM1 Antibody with antibodies against potential interacting partners

  • Quantify interaction signals in different cell types or tissues

Research has demonstrated that specific fragments of ECM1, particularly the δF/COOH (360–540 aa) and partial C δP/COOH (360–480 aa) terminals, can be used in binding studies to identify interaction partners .

What is the role of ECM1 in cardiac aging and how can it be investigated?

ECM1's role in cardiac aging represents an emerging research area:

• Expression analysis:

  • ECM1 mRNA and protein (~75kDa) are upregulated in aging cardiac tissue

  • ECM1 is specifically upregulated in the infarct zone of day-3 post-MI LV tissue

  • Mouse primary cultured cardiac fibroblasts (CFbs) show no ECM1 expression under control conditions, nor in response to TGF-β1 or ANG-II treatment

• Investigative approaches:

  • Flow cytometry can distinguish ECM1+ and ECM1- cardiac cell populations

  • tSNE analysis helps identify differential marker expression between these populations

  • Histograms can visualize the differential expression patterns

• Functional studies:

  • Compare cardiac function parameters between wild-type and ECM1-knockout models

  • Assess fibrosis development and inflammatory responses

  • Evaluate response to cardiac stress (e.g., ischemia-reperfusion)

This emerging field offers opportunities to connect ECM1's known roles in inflammation and tissue remodeling with age-related cardiac pathologies.

The upregulation of ECM1 in post-MI cardiac tissue suggests a potential role in the response to cardiac injury, though the precise mechanisms remain to be fully elucidated .