BVES Antibody

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

BVES (Blood Vessel Epicardial Substance), also known as POPDC1 (Popeye domain-containing protein 1), is a transmembrane protein that plays critical roles in cell adhesion and maintenance of cell integrity. It is involved in the formation and regulation of tight junction paracellular permeability barriers in epithelial cells . BVES is expressed in various tissues derived from all three germ layers, with particularly high expression in cardiac and skeletal muscle . Recent research has revealed its involvement in muscular dystrophy, cardiac arrhythmia, and cancer, making it an important target for both basic science and translational research .

What types of BVES antibodies are available for research?

Both monoclonal and polyclonal antibodies targeting BVES are commercially available. Monoclonal antibodies offer high specificity for particular epitopes, while polyclonal antibodies recognize multiple epitopes, potentially providing stronger signals. For instance:

Researchers should select antibodies based on their specific experimental needs, considering factors such as species reactivity, applications, and target regions .

What are the validated applications for BVES antibodies?

BVES antibodies have been validated for multiple applications:

• Western Blotting (WB): Detecting BVES protein (~41 kDa) in tissue lysates and cell lines

• Immunohistochemistry (IHC): Examining BVES expression in paraffin-embedded tissue sections

• Immunofluorescence (IF): Analyzing subcellular localization in cultured cells and tissue sections

• Affinity chromatography: Purifying BVES protein from biological samples

• Functional studies: Blocking antibodies can be used to study BVES function in processes like cellular migration

Each application requires specific optimization regarding antibody concentration, incubation conditions, and detection methods .

How should I choose between monoclonal and polyclonal BVES antibodies for my specific research question?

The choice depends on your experimental goals:

For detecting subcellular localization patterns: Consider using both types, as they may reveal different distribution patterns. Smith et al. (2006) demonstrated that monoclonal antibodies (SB series) show broader distribution along the lateral membrane, while polyclonal antibodies (B846) recognize BVES primarily at points of cell-cell contact . This difference may reflect epitope accessibility or different Bves isoforms.

For specificity verification: Use monoclonal antibodies that have been specifically tested against other Popdc family members (Popdc2, Popdc3) to ensure no cross-reactivity . The SB1 monoclonal antibody has been rigorously validated and shown to react specifically with Bves but not with Popdc2 or Popdc3 in immunoblotting assays .

For detection in multiple species: Check the species reactivity data. Many antibodies work across human, mouse, and rat samples due to high conservation of BVES protein sequence .

What are the optimal fixation and antigen retrieval methods for BVES immunohistochemistry?

Based on published protocols:

Fixation: Most successful protocols use cold 70% methanol fixation for 10 minutes for cultured cells and tissue sections . For paraffin-embedded sections, standard formalin fixation works well when combined with appropriate antigen retrieval.

Antigen retrieval: For paraffin sections:

• Heat-induced epitope retrieval using citrate buffer (pH 6.0) is suitable for many BVES antibodies

• Some antibodies perform better with TE buffer at pH 9.0

• For the Human Protein Atlas antibodies, specific protocols are provided that optimize signal-to-noise ratio

Permeabilization: Using 0.25% Triton X-100 in PBS for 10 minutes following fixation enhances antibody accessibility to intracellular epitopes .

After these steps, blocking with 2% bovine serum albumin in PBS for one hour at room temperature is recommended to reduce non-specific binding .

How can I validate the specificity of my BVES antibody?

Multiple validation approaches should be employed:

• Expression systems verification: Test antibody reactivity on cells transfected with BVES expression constructs compared to empty vector controls . The antibody should detect overexpressed BVES with appropriate molecular weight and subcellular localization.

• Cross-reactivity testing: Verify absence of signal when testing against related family members (e.g., Popdc2, Popdc3) using GST-fusion proteins of each Popdc family member in immunoblotting assays .

• Western blot analysis: Confirm detection of a band at the expected molecular weight (~41 kDa) in tissues known to express BVES (heart, skeletal muscle) .

• Cell line panel: Test antibody on cell lines with documented BVES expression like EMC (rat epithelial epicardium), C2C12 (mouse skeletal myoblasts), HCE (human corneal epithelium), and MDCK (canine kidney epithelial cells) .

• siRNA knockdown: For enhanced validation, demonstrate reduced antibody signal following BVES gene silencing .

How can BVES antibodies be used to study cell-cell adhesion mechanisms?

BVES localizes to points of cell-cell contact and precedes classical junctional markers during junction formation, making it valuable for studying early adhesion events . Methodological approaches include:

• Time-course analysis: Use BVES antibodies in conjunction with markers for tight junctions (ZO-1, Occludin) and adherens junctions (E-cadherin) to track the sequence of protein recruitment during junction formation . This reveals that BVES is one of the first proteins transported to the membrane as cells begin to adhere .

• Calcium switch assays: Monitor BVES localization during calcium depletion and repletion to study dynamic redistribution during junction disruption and reformation .

• Cell reaggregation assays: Track BVES localization at nascent points of cell-cell contact in reaggregating myocytes to analyze its role in initial cell coupling .

• Blocking antibody experiments: Apply antibodies targeting the extracellular domain of BVES (e.g., amino acids 15-37) to confluent cell monolayers to study the functional importance of BVES in maintaining cell adhesion. Chang and Russ demonstrated that such treatment leads to cell membrane dehiscence while adhesion junction proteins remain at the membrane .

How do different BVES antibodies reveal distinct subcellular localization patterns?

Interestingly, different antibodies against BVES reveal varying distribution patterns, providing insight into protein function:

• Polyclonal antibody B846: Recognizes BVES primarily at tight junctions and points of cell-cell contact . In cardiac myocytes, B846 detects BVES only at intercalated discs (cell-cell contact points) .

• Monoclonal antibodies (SB series): Show broader distribution along the lateral membrane of epithelial cells, co-localizing with both tight junction markers (ZO-1, Occludin) and adherens junction proteins (E-cadherin) .

• Subcellular dynamics: Both antibody types reveal that BVES exhibits a dynamic localization pattern. When cells are not in contact, BVES is observed intracellularly. As cells begin to adhere, BVES rapidly translocates to the membrane, preceding classical junctional markers .

These differential patterns may reflect:

• Epitope masking in certain subcellular contexts

• Differential recognition of BVES conformational states

• Detection of distinct BVES isoforms with different functional roles

What methodological approaches can resolve contradictory BVES expression data between mRNA and protein detection methods?

Discrepancies exist between BVES expression patterns detected by in situ hybridization versus immunochemical methods, particularly regarding epithelial expression . To resolve these contradictions:

• Multi-method validation: Combine RT-qPCR, in situ hybridization, and immunodetection using multiple antibodies targeting different epitopes .

• Single-cell analysis: Employ single-cell RNA sequencing alongside immunofluorescence to correlate transcript and protein levels at the individual cell level .

• Knockout/knockdown controls: Include BVES knockout or knockdown samples as negative controls to validate antibody specificity and rule out non-specific binding .

• Species-comparative approach: Compare expression across multiple species using the same detection methods, as conserved patterns are more likely to reflect true biological expression .

• Sensitivity enhancement: For mRNA detection, consider using RNAscope or other amplification methods if BVES transcript levels are low in certain tissues .

The discrepancy may be due to low levels of BVES mRNA in non-muscle cell types, making detection by in situ hybridization difficult despite protein presence .

What are the common pitfalls when working with BVES antibodies in Western blot applications?

Several challenges may arise:

• Variable molecular weight detection: BVES can appear between 41-70 kDa on Western blots due to post-translational modifications and splice variants . Use positive controls (heart or skeletal muscle tissue) to establish expected band patterns .

• Sample preparation: BVES is a transmembrane protein requiring proper extraction. Use buffers containing:

  • 1% Igepal CA-630 (or NP-40)

  • 0.5% sodium deoxycholate

  • 0.1% SDS

  • Protease inhibitor cocktail

  Homogenize tissue samples thoroughly and centrifuge at 21,000 × g for 30 minutes at 4°C .

• Transfer conditions: BVES may require optimized transfer conditions for efficient membrane binding. Use PVDF membranes (Immobilon-P) rather than nitrocellulose for better protein retention .

• Blocking optimization: Use 10% nonfat dry milk in TBST (100mM Tris-Cl pH 7.5, 150 mM NaCl, 0.25% TritonX-100) for blocking to reduce background .

• Antibody concentration: Titrate primary antibody concentrations carefully. For monoclonal antibodies like SB1, a 1:2000 dilution (~1 μg/mL) is typically effective .

How can I optimize BVES immunofluorescence staining to detect different subcellular pools?

BVES shows dynamic subcellular localization depending on cell status:

• Fixation method comparison: Compare different fixation methods:

  • Cold 70% methanol (10 minutes) works well for membrane-localized BVES

  • 4% paraformaldehyde may better preserve intracellular structures to detect cytoplasmic BVES pools

• Permeabilization optimization: Test different permeabilization conditions:

  • 0.25% Triton X-100 (10 minutes) for complete membrane permeabilization

  • 0.1% saponin for milder permeabilization that better preserves membrane structures

• Detection of dynamic changes: To capture the dynamic redistribution of BVES:

  • Examine cells at different densities and time points after plating

  • In long-term confluent cultures, BVES localizes almost exclusively at the cell surface

  • In non-contacting cells, BVES shows primarily intracellular localization

• Confocal Z-stack analysis: Perform thorough Z-stack imaging to distinguish between apical, lateral, and basal distributions of BVES in polarized epithelial cells .

• Co-staining strategies: Combine BVES antibodies with markers for:

  • Tight junctions (ZO-1, Occludin)

  • Adherens junctions (E-cadherin, β-catenin)

  • Vesicular trafficking (VAMP3)

  • Caveolae (Caveolin-1)

What controls should be included when using BVES antibodies for functional blocking experiments?

When using antibodies to block BVES function (as in Chang and Russ's study ):

• Antibody targeting controls:

  • Include antibodies targeting intracellular domains of BVES as negative controls (they should not affect function since they cannot access the target in intact cells)

  • Use isotype-matched control antibodies at equivalent concentrations to control for non-specific effects

  • Test a concentration gradient (e.g., 0.1-1.0 ng/mL) to establish dose-dependent effects

• Functional readouts:

  • Measure transepithelial electrical resistance (TER) to assess barrier function changes

  • Perform scratch/wounding assays to quantify effects on collective cell migration

  • Analyze RhoA activation status to link functional changes to specific signaling pathways

• Complementary approaches:

  • Compare antibody blocking effects with siRNA knockdown

  • Rescue experiments using BVES constructs with mutations in the antibody-binding epitope

  • Use recombinant BVES extracellular domain as a competitive inhibitor to confirm specificity

How can BVES antibodies contribute to research on muscular dystrophy pathogenesis?

Recent research has identified BVES mutations as causal for limb-girdle muscular dystrophy type 25 . BVES antibodies can be invaluable in this research:

• Mechanism investigation: Use BVES antibodies to track protein expression and localization in:

  • Patient-derived muscle biopsies

  • Animal models with BVES knockout or mutation

  • Cell culture systems modeling the disease

• Protein interaction studies: Employ co-immunoprecipitation with BVES antibodies to identify disrupted protein interactions in diseased states. Recent studies show BVES interacts with ADCY9 and regulates cyclic AMP signaling .

• Therapeutic development assessment: Monitor BVES protein restoration in:

  • Gene therapy approaches using adeno-associated virus (AAV)-BVES

  • Pharmacological interventions like bortezomib (which ameliorates pathology in BVES-knockout skeletal muscle)

• Biomarker development: Assess whether BVES antibodies can detect disease-specific changes in protein expression, localization, or post-translational modifications that could serve as diagnostic or prognostic biomarkers .

What methodological approaches can resolve the differential cellular localization patterns detected by distinct BVES antibodies?

The discrepancy between different antibodies' staining patterns provides an interesting research question:

• Epitope mapping: Precisely map the binding epitopes of different antibodies using:

  • Peptide arrays covering the entire BVES sequence

  • Competitive binding assays with defined peptide fragments

  • Hydrogen-deuterium exchange mass spectrometry to identify antibody binding regions

• Conformation-specific detection: Test whether antibodies recognize different conformational states of BVES using:

  • Native versus denatured protein samples

  • Cross-linking studies to stabilize specific conformations

  • Mutagenesis of potential regulatory sites that might affect conformation

• Post-translational modification analysis: Investigate whether modifications affect antibody recognition:

  • Phosphorylation state analysis (BVES contains multiple potential phosphorylation sites)

  • Glycosylation analysis

  • Ubiquitination detection

• Super-resolution microscopy: Employ techniques like STORM or PALM with differently labeled antibodies to precisely map spatial relationships between epitopes at nanometer resolution .

How can BVES antibodies be utilized to investigate its role in cancer progression?

BVES has been implicated in several cancer types, including non-small-cell lung cancer and colorectal cancer . Antibody-based approaches include:

• Expression profiling: Use BVES antibodies for:

  • Tissue microarray analysis across cancer types and stages

  • Correlation of expression with clinical outcomes

  • Comparative analysis between tumor and adjacent normal tissue

• Signaling pathway analysis: Study BVES's role in cancer-relevant signaling:

  • RhoA pathway regulation (BVES modulates RhoA activity)

  • Cell adhesion signaling (BVES affects tight junction formation)

  • Metastatic potential (BVES regulates cell motility and migration)

• Functional studies: Apply BVES antibodies to:

  • Block BVES function in 3D cell culture models to assess impacts on tumor spheroid formation

  • Examine effects on epithelial-mesenchymal transition markers

  • Investigate cell invasion and migration in transwell assays

• Therapeutic potential: Explore whether:

  • BVES-targeting antibodies affect cancer cell viability or invasiveness

  • Antibody-drug conjugates directed against BVES show anti-tumor activity

  • BVES expression level correlates with response to specific cancer therapies