MUC20 Antibody

What is the biological significance of MUC20 in cancer research?

At the molecular level, MUC20 regulates the MET signaling cascade by decreasing hepatocyte growth factor (HGF)-induced MAPK activation. It functions by blocking GRB2 recruitment to MET, thus suppressing the GRB2-RAS pathway and inhibiting HGF-induced proliferation of MMP1 and MMP9 expression .

How do I select the appropriate MUC20 antibody for my experimental needs?

Selection of the appropriate MUC20 antibody depends on several experimental factors:

For Western blotting, the observed molecular weight of MUC20 ranges from 70-80 kDa, which may differ from the calculated weight due to post-translational modifications . Several validated commercial antibodies target different regions of MUC20, including AA 100-200, AA 113-347, and the C-terminus .

How can I validate the specificity of MUC20 antibodies in my experimental system?

Validation of MUC20 antibody specificity is critical for obtaining reliable results. Multiple approaches should be employed:

• Knockdown experiments: The gold standard for antibody validation involves depleting the target protein using siRNA or shRNA. In previous studies, depletion of MUC20 in stratified HCLE cells by siRNA demonstrated antibody specificity .

• Overexpression systems: Transfecting cell lines with MUC20 expression vectors serves as a positive control. Western blot analysis can compare transfected versus non-transfected lysates, as demonstrated in studies using MUC20-transfected 293T cells that showed a distinct band at approximately 80 kDa compared to non-transfected controls .

• Immunodetection controls: Include both positive and negative controls:

  • Positive controls: Kidney tissue expresses high levels of MUC20, particularly in proximal tubules

  • Negative controls: Omit primary antibody in parallel experiments

  • Peptide competition assays: Pre-incubate antibody with immunizing peptide to confirm binding specificity

• Multiple antibody comparison: Use antibodies targeting different epitopes (N-terminus vs. C-terminus) to confirm consistent detection patterns .

What are the optimal protocols for detecting MUC20 in tissue samples?

Optimized protocols for MUC20 detection vary by application:

Immunohistochemistry (IHC) Protocol:

• For frozen sections (7-μm), fix with methanol

• Block with 3% bovine serum albumin in PBS

• Incubate with primary anti-MUC20 antibody (typically 1:50-1:150 dilution) overnight at 4°C

• Apply appropriate secondary antibody (1:300 dilution) for 1 hour at room temperature

• Mount using medium containing DAPI for nuclear counterstaining

Western Blot Protocol:

• Resolve proteins in 10% SDS-PAGE and electroblot onto nitrocellulose membranes

• Block with 5% nonfat milk in 0.1% Tween 20 in Tris-buffered saline (TTBS) for 1 hour

• Incubate with anti-MUC20 antibody (typically 1:500-1:3,000 dilution) overnight at 4°C

• Apply corresponding peroxidase-conjugated secondary antibody (1:5,000)

• Visualize using chemiluminescence detection

For challenging samples, optimization of antigen retrieval methods may be necessary. The recommended antibody dilutions range from 1:500-1:2000 for Western blotting and 1:50-1:500 for immunofluorescence/immunocytochemistry .

How does MUC20 expression correlate with tumor immune microenvironment?

Recent research has revealed complex interactions between MUC20 expression and tumor-infiltrating immune cells (TICs). In ccRCC, MUC20 expression has been shown to correlate with specific immune cell populations:

These correlations suggest MUC20 may influence the immune component of the tumor microenvironment . Gene Set Enrichment Analysis has shown that low MUC20 expression groups are primarily enriched in immune-related activities, inflammation, and epithelial-mesenchymal transition . This indicates that MUC20 may serve as a biomarker for immunotherapy response, with patients having low MUC20 expression potentially showing better responses to immune checkpoint blockades (ICBs) .

What approaches can I use to investigate MUC20's role in cancer progression?

To investigate MUC20's functional role in cancer progression, multiple experimental approaches can be employed:

• Genetic manipulation studies:

  • Knockdown using shRNA or siRNA: In previous studies, transfection of pGPU6/GFP/Neo-shRNA-MUC20 reduced MUC20 expression and significantly decreased invasive ability of colorectal cancer cells

  • Overexpression systems: Transfection with pIRES2-EGFP-MUC20 has been shown to enhance migration and invasion abilities of CRC cells

• Functional assays:

  • Wound healing assays to assess cell migration

  • Matrigel Transwell invasion assays to quantify invasive potential

  • Western blotting to identify downstream signaling molecules affected by MUC20 (e.g., MMP-2, MMP-3, E-cadherin)

• In vivo models:

  • Xenograft models with MUC20-manipulated cancer cells to assess tumor growth and metastasis

  • Patient-derived xenografts to evaluate MUC20 as a prognostic marker

  • Analysis of MUC20 correlation with treatment response, particularly to immune checkpoint inhibitors

• Mechanistic studies:

  • Investigation of MUC20's interaction with MET signaling pathway

  • Analysis of MUC20's effect on HGF-induced MAPK activation

  • Evaluation of MUC20's role in modulating tumor microenvironment

How can I optimize MUC20 co-localization studies with other proteins?

For effective co-localization studies of MUC20 with other proteins:

• Sample preparation:

  • Use fresh frozen tissue when possible, as it better preserves epitope accessibility

  • If using FFPE tissues, optimize antigen retrieval methods (heat-induced epitope retrieval in citrate buffer is often effective)

• Double immunolabeling protocol:

  • Apply a mixture of primary antibodies (e.g., rabbit anti-MUC20 C-terminus [1:50] and mouse antibody against your protein of interest [1:50])

  • Follow with a mixture of differentially labeled secondary antibodies (e.g., FITC-conjugated anti-rabbit [1:300] and Texas red-conjugated anti-mouse [1:300])

  • Include appropriate controls (single antibody staining, isotype controls)

• Imaging considerations:

  • Use confocal microscopy for precise co-localization analysis

  • Perform Z-stack imaging to evaluate co-localization in multiple planes

  • Use appropriate software for quantitative co-localization analysis (e.g., ImageJ with co-localization plugins)

Previous studies have successfully used this approach to demonstrate MUC20 co-localization with MUC5AC in conjunctival tissue .

What are common technical challenges when working with MUC20 antibodies?

How has MUC20 been implicated in colorectal cancer progression?

MUC20 plays a significant role in colorectal cancer progression through multiple mechanisms:

What is known about MUC20's function in clear cell renal cell carcinoma (ccRCC)?

In ccRCC, MUC20 has emerged as a novel prognostic biomarker with immune-related functions:

• Correlation with survival: MUC20 expression is positively correlated with survival rates in ccRCC patients .

• Association with clinicopathologic characteristics: MUC20 expression negatively correlates with adverse clinicopathologic characteristics, including higher grade, advanced clinical and TNM stages .

• Immune microenvironment modulation: MUC20 expression correlates with specific tumor-infiltrating immune cell populations, suggesting a role in shaping the immune landscape within tumors. Specifically, MUC20 positively correlates with CD8+ T cells and resting mast cells, while negatively correlating with activated CD4+ memory T cells, Treg cells, and plasma cells .

• Immunotherapy response prediction: Low MUC20 expression is associated with enrichment of immune-related activities, suggesting patients with low MUC20 expression may have better responses to immune checkpoint blockade therapies .

• Potential therapeutic implications: Analysis of pharmaceutical sensitivity indicates that 17 potential anticancer drugs might be particularly effective in patients with different MUC20 expression levels, opening avenues for personalized medicine approaches .

How can researchers assess MUC20's role in signaling pathways?

To investigate MUC20's involvement in signaling pathways, particularly the MET/HGF axis:

• Protein-protein interaction studies:

  • Co-immunoprecipitation to detect MUC20 binding partners

  • Proximity ligation assays to visualize protein interactions in situ

  • FRET/BRET assays for real-time monitoring of protein-protein interactions

• Pathway activation analysis:

  • Western blotting to assess phosphorylation status of downstream effectors (MAPK, AKT)

  • Reporter gene assays to measure pathway activity

  • Phospho-kinase arrays to evaluate multiple signaling nodes simultaneously

• Functional response to pathway modulation:

  • HGF stimulation experiments in MUC20-expressing versus MUC20-depleted cells

  • Combination of MUC20 manipulation with pathway inhibitors (e.g., c-Met inhibitors)

  • Assessment of cellular responses (proliferation, migration, invasion) following pathway perturbation

• Expression correlation analysis:

  • Bioinformatic analysis of gene expression datasets to identify correlations between MUC20 and signaling pathway components

  • Single-cell RNA sequencing to determine cell-specific co-expression patterns

Research has shown that MUC20 expression is inversely related to activation of c-Met and downstream p44/42 MAPK pathway. Mechanistically, c-Met activation with hepatocyte growth factor (HGF) can induce proteasome inhibitor resistance, while c-Met inhibition restores sensitivity .

What emerging technologies might enhance MUC20 research?

Several cutting-edge technologies offer promise for advancing MUC20 research:

• Single-cell analysis: Single-cell RNA sequencing and mass cytometry can reveal heterogeneity in MUC20 expression and its correlation with cell states within tumors and normal tissues.

• CRISPR/Cas9 gene editing: Precise genome editing allows creation of MUC20 knockout or knock-in models to study its function in relevant biological contexts.

• Patient-derived organoids: Three-dimensional culture systems derived from patient samples provide physiologically relevant models to study MUC20's role in disease progression and treatment response.

• Spatial transcriptomics: These techniques enable visualization of MUC20 expression patterns within the tissue microenvironment, providing insights into its spatial relationship with immune cells and stromal components.

• Proteomics approaches: Advanced mass spectrometry methods can identify post-translational modifications of MUC20 and map its interactome in different cellular contexts.

• In vivo imaging: Development of MUC20-targeted probes for molecular imaging could enable non-invasive monitoring of MUC20 expression in animal models and potentially in patients.

• Artificial intelligence: Machine learning approaches can integrate multi-omics data to predict MUC20's functional impact and potential as a biomarker across various cancer types.

How might MUC20 research translate to clinical applications?

The current body of MUC20 research suggests several potential clinical applications:

• Prognostic biomarker development: MUC20 overexpression has been identified as an independent prognostic factor in colorectal cancer, suggesting its potential as a clinical biomarker for risk stratification .

• Therapeutic target identification: MUC20's role in promoting migration and invasion of cancer cells makes it a potential therapeutic target. Inhibiting MUC20 expression or function could potentially reduce cancer aggressiveness .

• Predictive biomarker for immunotherapy: MUC20 expression correlates with tumor-infiltrating immune cell composition, suggesting its potential as a predictive biomarker for response to immune checkpoint inhibitors in ccRCC .

• Patient stratification for targeted therapies: The correlation between MUC20 expression and sensitivity to specific anticancer drugs suggests its utility in guiding treatment selection .

• Monitoring disease progression: Serial measurement of MUC20 expression in liquid biopsies could potentially serve as a non-invasive method for monitoring disease status and treatment response.

• Combination therapy design: Understanding MUC20's role in signaling pathways, particularly its interaction with the MET/HGF axis, could inform the development of rational combination therapies targeting these pathways .