MARVELD3 Antibody

What is MARVELD3 and why are specific antibodies important for its study?

MARVELD3 is a member of the Tight junction Associated Marvel Proteins (TAMP) family, which includes occludin and tricellulin. It functions as a novel integral membrane component of epithelial junctions . MARVELD3 exists in two isoforms (splice variants) that share an N-terminal cytoplasmic domain of 198 amino acids but differ in their C-terminal halves containing the transmembrane domains . Unlike other TAMP members, both MARVELD3 isoforms possess relatively short C-terminal cytoplasmic domains (30aa for isoform 1; 18aa for isoform 2) .

Specific antibodies against MARVELD3 are crucial for investigating its expression, localization, and function in various tissues and disease states. When selecting an antibody, researchers should consider the specific amino acid regions targeted and whether the antibody can distinguish between isoforms. Many commercial MARVELD3 antibodies target the shared N-terminal domain (AA 25-115), allowing detection of both isoforms simultaneously .

Which experimental applications are most suitable for MARVELD3 antibodies?

MARVELD3 antibodies have been validated for several experimental applications, with varying effectiveness depending on the specific antibody and target species. The most commonly employed techniques include:

• Western Blotting (WB): Effective for detecting MARVELD3 protein expression levels in cell or tissue lysates. The protein typically appears as a band of approximately 40 kDa .

• ELISA: Useful for quantitative measurement of MARVELD3 in solution.

• Immunofluorescence (IF): Enables visualization of MARVELD3 localization at cellular junctions.

• Immunohistochemistry (IHC): Allows detection of MARVELD3 in fixed tissue sections.

When designing experiments, researchers should verify the antibody's validated applications and optimize protocols accordingly. For instance, in immunofluorescence studies of epithelial cells, co-staining with established tight junction markers like occludin can help confirm specific junctional localization of MARVELD3 .

How should researchers approach MARVELD3 antibody validation?

Proper validation of MARVELD3 antibodies is essential to ensure experimental reproducibility and accurate data interpretation. A comprehensive validation approach should include:

• Specificity testing: Compare signals in control samples versus those with known MARVELD3 expression manipulation (knockdown/overexpression).

• Western blot analysis: Confirm detection of a single band at the expected molecular weight (~40 kDa).

• siRNA knockdown controls: Use siRNA targeting MARVELD3 to demonstrate antibody specificity. Research has shown that efficient knockdown can be achieved with pooled siRNAs or individual sequences (e.g., siRNAs 14 and 16 targeting the common N-terminal domain) .

• Overexpression confirmation: Transfect cells with cDNAs encoding MARVELD3 isoforms and verify increased signal intensity.

When validating antibodies against MARVELD3, researchers should be aware that some antibodies may only recognize specific species (e.g., human) or particular isoforms . Always include appropriate positive and negative controls in validation experiments.

How can MARVELD3 antibodies be utilized to investigate tight junction assembly and maintenance?

MARVELD3 antibodies provide valuable tools for investigating tight junction (TJ) biology through both microscopic and biochemical approaches. To effectively study TJ assembly and maintenance:

• Live-cell imaging: Use fluorescently-labeled MARVELD3 antibodies to track dynamic changes in TJ composition during junction formation in epithelial monolayers.

• Co-immunoprecipitation: Employ MARVELD3 antibodies to identify protein-protein interactions within the junctional complex.

• Functional knockdown studies: Combine siRNA knockdown with MARVELD3 antibody staining to assess how depletion affects localization and expression of other junctional proteins .

Research has shown that efficient MARVELD3 knockdown results in its absence from junctional staining by immunofluorescence, confirming the utility of these antibodies for monitoring junctional pools of MARVELD3 . When designing such experiments, controls should include assessment of other junctional proteins to distinguish between specific effects on MARVELD3 and general disruption of junctional integrity.

What methodological considerations are important when using MARVELD3 antibodies for cancer research?

MARVELD3 has emerged as a potential prognostic biomarker in cancer research, particularly in oral squamous cell carcinoma (OSCC) where its high expression correlates with poor prognosis . When using MARVELD3 antibodies for cancer research:

• Compare expression levels: Analyze MARVELD3 expression in tumor versus matched normal tissues using appropriately calibrated immunoblotting or immunohistochemistry.

• Correlate with clinical outcomes: Combine antibody-based detection with patient survival analysis to establish prognostic significance.

• Evaluate relationship with other biomarkers: Use multiplex staining to assess co-expression with established cancer markers.

How can researchers investigate the relationship between MARVELD3 and immune cell infiltration using antibodies?

The relationship between MARVELD3 expression and immune cell infiltration represents an emerging area of research, particularly in cancer immunology. To investigate this relationship:

• Multiplex immunofluorescence: Combine MARVELD3 antibodies with markers for specific immune cell populations (e.g., CD3 for T cells, CD20 for B cells).

• Tissue microarray analysis: Use MARVELD3 antibodies on cancer tissue microarrays with parallel staining for immune markers.

• Flow cytometry: For dissociated tissues, use MARVELD3 antibodies in conjunction with immune cell markers to correlate expression with specific infiltrating populations.

Research has demonstrated an inverse correlation between MARVELD3 expression and the infiltration of various immune cells, including dendritic cells, T cells, neutrophils, and B cells in OSCC . This suggests that high MARVELD3 expression may be associated with reduced immune surveillance. When designing such studies, researchers should consider both the spatial distribution of immune cells relative to MARVELD3-expressing cells and the quantitative relationship between expression levels and immune infiltrate density.

What are common challenges in Western blotting with MARVELD3 antibodies and how can they be addressed?

Western blotting for MARVELD3 can present several technical challenges that researchers should anticipate and address:

• Multiple bands: MARVELD3 exists as two isoforms and may show post-translational modifications, potentially resulting in multiple bands. Researchers should determine which bands represent specific MARVELD3 isoforms through validation with positive controls and knockdown experiments .

• Low signal strength: MARVELD3 may be expressed at relatively low levels in some tissues. Optimization strategies include:

  • Increasing protein concentration in samples

  • Extended primary antibody incubation (overnight at 4°C)

  • Using enhanced chemiluminescence detection systems

• Non-specific binding: Some antibodies may show cross-reactivity with other proteins. Researchers should:

  • Increase blocking time and concentration

  • Optimize antibody dilution through titration experiments

  • Consider using protein G-purified antibodies, which have shown >95% purity in some commercial preparations

When troubleshooting Western blot issues, always include positive controls (e.g., cells transfected with MARVELD3 cDNA) and negative controls (e.g., cells treated with MARVELD3 siRNA) to help distinguish specific from non-specific signals .

How should researchers address potential cross-reactivity concerns with MARVELD3 antibodies?

Cross-reactivity is a significant concern when working with antibodies against tight junction proteins, which may share structural similarities. To address potential cross-reactivity with MARVELD3 antibodies:

• Epitope mapping: Select antibodies targeting unique regions of MARVELD3 not conserved in other TAMP family members.

• Cross-adsorption controls: Pre-adsorb the antibody with recombinant proteins of related family members to reduce non-specific binding.

• Validation in knockout/knockdown systems: Confirm antibody specificity using genetic approaches to deplete MARVELD3.

What methodologies can researchers employ to study MARVELD3 isoform-specific expression and function?

MARVELD3 exists as two splice variants with different C-terminal domains, potentially conferring distinct functions. To investigate isoform-specific biology:

• RT-PCR analysis: Design primers specific to unique regions of each isoform for quantitative expression analysis across tissues and cell types.

• Isoform-specific antibodies: Utilize antibodies targeting the unique C-terminal regions of each isoform.

• Isoform-selective knockdown: Design siRNAs targeting unique exons to selectively deplete individual isoforms.

Research has demonstrated that both MARVELD3 isoforms are widely expressed in different epithelial and endothelial cells, though with tissue-specific differences. For example, liver and the hepatocyte-derived cell line HepG2 predominantly express isoform 1 . When designing isoform-specific studies, researchers should first characterize the isoform expression profile in their experimental system using RT-PCR before proceeding with functional studies.

How can researchers investigate the relationship between MARVELD3 expression and epigenetic regulation?

The relationship between MARVELD3 expression and epigenetic regulation, particularly DNA methylation, represents an important area of investigation in cancer research. To study this relationship:

• Methylation-specific PCR: Analyze the methylation status of MARVELD3 promoter regions.

• Bisulfite sequencing: Perform detailed analysis of specific CpG sites in regulatory regions.

• Correlation analysis: Compare methylation data with protein expression levels determined by antibody-based methods.

Research has demonstrated an inverse correlation between MARVELD3 expression and DNA methylation sites in oral squamous cell carcinoma, consistent with characteristics of oncogenes . This suggests that hypomethylation may contribute to MARVELD3 overexpression in cancer. When investigating epigenetic regulation, researchers should combine methylation analysis with chromatin immunoprecipitation (ChIP) studies to comprehensively characterize regulatory mechanisms.

How can researchers utilize MARVELD3 antibodies to investigate its potential as a cancer biomarker?

The potential of MARVELD3 as a cancer biomarker, particularly in oral squamous cell carcinoma, represents an emerging research direction. To investigate this potential:

• Tissue microarray analysis: Use validated MARVELD3 antibodies to screen large cohorts of cancer samples.

• Survival correlation: Combine expression data with clinical outcomes to establish prognostic value.

• Multivariate analysis: Integrate MARVELD3 expression with other clinical parameters to develop comprehensive prognostic models.

What experimental approaches can elucidate the functional significance of MARVELD3 in the protein-protein interaction network of tight junctions?

Understanding MARVELD3's interactions within the tight junction protein network requires sophisticated experimental approaches:

• Proximity ligation assays: Detect protein-protein interactions within intact junctions using antibody pairs.

• BioID or APEX proximity labeling: Identify proteins in close proximity to MARVELD3 in living cells.

• Protein-protein interaction (PPI) network analysis: Integrate experimental data with bioinformatic predictions.

Research has identified several proteins that interact with MARVELD3 through PPI network analysis . These interactions may provide insights into MARVELD3's functional roles beyond structural maintenance of tight junctions. When investigating protein interactions, researchers should combine multiple methodologies and validate key interactions through orthogonal approaches.