MYO7B Antibody

What is MYO7B and why is it significant in research contexts?

MYO7B (myosin VIIB) is an unconventional myosin with a molecular weight of approximately 242 kDa that functions as an actin-based motor protein . It contains a highly conserved N-terminal motor domain with ATP and actin binding sites, a central 'neck' region with at least five IQ motifs, and a C-terminal tail consisting of tandem MyTH4-FERM domains separated by an SH3 domain .

MYO7B is particularly significant because it:

• Acts as a critical component of the intermicrovillar adhesion complex (IMAC)

• Localizes specifically to the distal tips of microvilli in intestinal epithelial cells and kidney tissue

• Plays an essential role in microvillar organization and brush border assembly

• Mediates a specialized endocytosis pathway involved in cellular entry of cargos such as α-synuclein preformed fibrils (α-Syn PFFs)

This multifunctionality makes MYO7B antibodies valuable tools for studying cellular organization, protein trafficking, and potential pathological mechanisms in neurodegenerative diseases.

What are the primary experimental applications for MYO7B antibodies?

MYO7B antibodies are versatile research tools with applications across multiple methodologies:

MYO7B antibodies have been successfully employed to:

• Map the precise localization of MYO7B along the microvillar axis in epithelial cells

• Investigate the functional consequences of MYO7B knockdown in cell lines

• Study the role of MYO7B in endocytosis mechanisms

• Examine MYO7B expression across different tissues and cell types

What tissues and cell types should be used as positive controls for MYO7B antibody validation?

Based on documented expression patterns, the following tissues and cell lines serve as reliable positive controls for MYO7B antibody validation:

When validating a MYO7B antibody, researchers should observe:

• Enrichment at the distal portion of microvilli (L_PI = 0.83 ± 0.11 and 0.87 ± 0.11 in intestine and kidney, respectively, where 0 = base and 1 = tip)

• A calculated molecular weight of approximately 242 kDa on western blots

• Specific immunoreactivity that can be abolished in knockdown/knockout controls

What are the optimal sample preparation protocols for MYO7B immunohistochemistry?

For optimal MYO7B immunohistochemistry results:

Tissue Processing:

• Paraffin embedding is suitable for preserving MYO7B epitopes

• Fresh-frozen sections may also be used but require careful fixation optimization

Antigen Retrieval:

• For optimal results, use TE buffer at pH 9.0 for antigen retrieval

• Alternative approach: citrate buffer at pH 6.0 may be used if TE buffer is unavailable

Antibody Incubation:

• Start with a 1:50-1:500 dilution range, optimizing based on signal-to-noise ratio

• Incubate at 4°C overnight for consistent results

• Use a validated blocking solution containing 1-5% BSA to minimize background

Controls:

• Include MYO7B knockdown/knockout tissues as negative controls

• Perform absorption controls using the immunizing peptide when available

• Include known positive tissues (small intestine or kidney) in each experiment

The localization pattern should show distinct enrichment at microvillar tips in polarized epithelial cells, which serves as an internal validation of specificity.

How can researchers effectively employ MYO7B antibodies to study its function in endocytosis pathways?

MYO7B has been identified as a critical component in specialized endocytosis pathways, particularly for heparan sulfate proteoglycan (HSPG)-dependent cargo internalization . To effectively study this function:

Experimental Approaches:

• Knockdown/Knockout Studies:

  • Generate MYO7B-depleted cell lines using siRNA, shRNA, or CRISPR-Cas9

  • Validate knockdown efficiency by western blot and immunostaining

  • Assess cargo uptake using fluorescently labeled ligands (e.g., α-Syn PFFs, GFP+)

• Live-Cell Imaging:

  • Express fluorescently tagged MYO7B constructs (e.g., EGFP-MYO7B)

  • Use total internal reflection fluorescence microscopy (TIRF) to monitor MYO7B dynamics at the plasma membrane

  • Co-express markers for clathrin-coated pits (e.g., mCherry-tagged clathrin light chain) to study colocalization

• Cargo Specificity Analysis:

  • Compare uptake of HSPG-dependent cargos (α-Syn PFFs, polycation-DNA complexes) versus independent cargos (transferrin, monomeric α-Syn)

  • Quantify internalization rates using flow cytometry or quantitative fluorescence microscopy

Key Considerations:

• MYO7B function appears specific to HSPG-dependent cargos

• MYO7B contributes to membrane dynamics at clathrin-enriched domains

• Both the motor domain and tail domains have distinct functions in cargo transport

By systematically manipulating MYO7B expression and activity, researchers can dissect its specific contributions to endocytic pathways.

How can motor domain mutations in MYO7B be utilized to dissect its functional mechanisms?

Motor domain mutations provide valuable tools for investigating the mechanistic basis of MYO7B function. Research has revealed that:

These mutational approaches have revealed that:

• Active ATPase cycling and normal actin binding are essential for MYO7B tip enrichment

• Motor activity per se may be partially dispensable for some aspects of MYO7B localization

• Mutations that lock MYO7B in a strongly bound state lead to aberrant stress fiber localization

When designing experiments with MYO7B motor mutants:

• Include wild-type controls expressed at comparable levels

• Quantify localization using the ratio of microvillar tip intensity to cytoplasmic intensity

• Categorize cells based on targeting efficiency (none, moderate, robust)

• Consider potential dominant-negative effects on endogenous MYO7B function

This approach enables detailed dissection of the mechanistic requirements for MYO7B function in diverse cellular contexts.

What methodological approaches can resolve contradictory findings regarding MYO7B's role in brush border assembly?

Research has revealed complex and sometimes contradictory findings regarding MYO7B's precise role in brush border assembly. To address these contradictions, consider the following methodological approaches:

1. Temporal Analysis:

• Examine MYO7B function at different stages of brush border development (early formation versus maintenance)

• Use time-course experiments with inducible knockdown systems to distinguish between roles in assembly versus stability

2. Quantitative Morphometric Analysis:

• Measure specific parameters of microvillar organization:

  • Clustering frequency and pattern (tipi-shaped clusters)

  • Microvillar length and diameter

  • Inter-microvillar spacing and regularity

  • Types of microvillar interactions (tip-to-tip, tip-to-base, base-to-base)

3. High-Resolution Imaging:

• Combine scanning electron microscopy (SEM) with confocal microscopy for comprehensive structural analysis

• Use super-resolution techniques to resolve IMAC component localization

• Implement live-cell imaging to capture dynamic aspects of microvillar organization

4. Context-Dependent Analysis:

• Compare MYO7B functions across different epithelial cell types (intestinal versus kidney)

• Assess differential requirements in various model systems (cell lines versus primary cultures)

• Examine potential compensatory mechanisms involving related myosins

5. Interaction Studies:

• Perform biochemical analysis of MYO7B's interactions with other IMAC components (CDHR2, CDHR5, USH1C)

• Investigate how MYO7B knockdown affects expression and localization of these binding partners

By implementing these comprehensive approaches, researchers can resolve apparent contradictions and develop a more nuanced understanding of MYO7B's multifaceted roles in brush border assembly and maintenance.

What considerations are critical when using MYO7B antibodies to study its role in endocytosis of α-synuclein fibrils and neurodegenerative disease models?

When investigating MYO7B's role in α-synuclein (α-Syn) fibril endocytosis and neurodegenerative disease processes, several critical considerations must be addressed:

1. Cell Type Specificity:

• Different cell types may exhibit varying dependencies on MYO7B-mediated endocytosis

• Compare findings between relevant cell models:

  • Neuronal cells (primary neurons, neuronal cell lines)

  • Non-neuronal cells (HEK293T, U2OS)

  • Neuron-glia co-cultures

2. Cargo Preparation and Characterization:

• α-Syn preformed fibrils (PFFs) must be consistently prepared and characterized:

  • Verify fibril formation by electron microscopy or thioflavin T binding

  • Ensure consistent fibril size distribution

  • Label fibrils appropriately (fluorescent tags should not disrupt uptake mechanisms)

3. Mechanistic Pathway Delineation:

• Distinguish MYO7B's role from other endocytic mechanisms:

  • Use parallel inhibitors of clathrin-mediated endocytosis (CME)

  • Assess HSPG dependency through enzymatic digestion or genetic approaches

  • Compare with other cargo types to establish specificity

4. Physiological Relevance Assessment:

• Connect MYO7B-mediated uptake to disease-relevant outcomes:

  • Monitor intracellular α-Syn aggregation and spreading

  • Assess neuronal toxicity markers

  • Evaluate modifications to standard protocols that better represent in vivo conditions

5. Therapeutic Implication Considerations:

• Determine if MYO7B represents a viable therapeutic target:

  • Test pharmacological inhibitors of myosin function

  • Identify specific domains that could be targeted without disrupting essential functions

  • Assess potential off-target effects on other myosin family members

These methodological considerations are essential for establishing robust connections between MYO7B function and neurodegenerative disease processes, potentially leading to novel therapeutic strategies for conditions like Parkinson's disease.

What are the most common technical challenges when using MYO7B antibodies and their solutions?

Researchers frequently encounter several technical challenges when working with MYO7B antibodies. Here are the most common issues and their methodological solutions:

1. High Background or Non-specific Staining:

• Problem: Diffuse staining throughout tissues or cells obscuring specific MYO7B signals

• Solutions:

  • Optimize blocking conditions (increase BSA percentage to 3-5%)

  • Extend blocking time (2-3 hours at room temperature)

  • Use species-specific serum matching secondary antibody host

  • Titrate primary antibody (try higher dilutions: 1:200-1:500)

  • Include 0.1-0.3% Triton X-100 in wash buffers to reduce non-specific binding

2. Weak or Absent Signal:

• Problem: Inability to detect MYO7B despite appropriate positive controls

• Solutions:

  • Optimize antigen retrieval (test both TE buffer pH 9.0 and citrate buffer pH 6.0)

  • Increase antibody concentration (try 1:50 dilution)

  • Extend primary antibody incubation time (overnight at 4°C)

  • Use signal amplification methods (tyramide signal amplification, polymer detection systems)

  • Confirm tissue fixation conditions (overfixation can mask epitopes)

3. Inconsistent Results Between Experiments:

• Problem: Variable staining patterns or intensities between replicate experiments

• Solutions:

  • Standardize all protocol parameters (fixation time, antigen retrieval duration, antibody dilutions)

  • Prepare larger volumes of antibody dilutions to use across multiple experiments

  • Include internal positive controls in each experiment (known positive tissues)

  • Use automated staining platforms when available

  • Aliquot antibodies to avoid freeze-thaw cycles

4. Discrepancies Between Antibody Lots:

• Problem: Different results obtained with new antibody lots

• Solutions:

  • Validate each new lot against previous successful lots

  • Maintain detailed records of lot numbers and their optimal conditions

  • Purchase larger quantities of validated lots when critical experiments are planned

  • Consider using antibodies with validation data across multiple applications

5. Detection of Multiple Bands in Western Blot:

• Problem: Additional bands besides the expected 242 kDa MYO7B band

• Solutions:

  • Optimize sample preparation (use protease inhibitors)

  • Adjust polyacrylamide percentage for better resolution of high molecular weight proteins

  • Perform absorption controls with immunizing peptide

  • Verify bands using MYO7B knockdown/knockout controls

  • Consider potential isoforms (up to 2 different isoforms have been reported)

Consistent application of these troubleshooting approaches will significantly improve the reliability of MYO7B antibody-based experiments.

What considerations are important when comparing results from different MYO7B antibodies in multilabel immunofluorescence studies?

When conducting multilabel immunofluorescence studies involving multiple MYO7B antibodies or combining MYO7B antibodies with antibodies against other proteins, several critical considerations must be addressed:

1. Epitope Overlap and Antibody Competition:

• Different MYO7B antibodies may target overlapping epitopes, leading to competitive binding

• Solutions:

  • Map epitope regions of each antibody (N-terminal motor domain vs. C-terminal tail)

  • Apply antibodies sequentially rather than simultaneously if targeting similar regions

  • Validate antibody compatibility in single-label controls before combining

2. Species Cross-Reactivity Management:

• When combining antibodies from the same host species:

  • Use directly conjugated primary antibodies to avoid secondary antibody cross-reactivity

  • Implement sequential staining with intermediate blocking steps

  • Consider Fab fragment blocking between sequential antibody applications

  • Validate signal separation using spectral unmixing on confocal systems

3. Signal Intensity Calibration:

• Different antibodies may produce varying signal intensities, complicating comparative analysis

• Approaches:

  • Establish internal references for normalization (e.g., ratio to a consistent cellular marker)

  • Optimize individual antibody dilutions to achieve comparable signal ranges

  • Use identical imaging parameters across comparison groups

  • Include fluorescence standards to calibrate absolute intensity values

4. Fixation and Antigen Retrieval Compatibility:

• Various antibodies may require different sample preparation methods

• Solutions:

  • Test compatibility of suggested protocols (e.g., TE buffer pH 9.0 vs. citrate buffer pH 6.0)

  • Determine compromise conditions that preserve epitopes for all antibodies

  • Consider multiple sequential staining protocols if conditions cannot be reconciled

  • Validate epitope preservation using known positive controls for each antibody

5. Validation Through Complementary Approaches:

• Confirm co-localization findings using independent methods:

  • Proximity ligation assay (PLA) to verify protein-protein interactions

  • Co-immunoprecipitation to biochemically validate interactions observed in imaging

  • Express fluorescently tagged proteins to confirm antibody-based localization patterns

  • Use super-resolution microscopy to resolve closely associated but distinct proteins

By systematically addressing these considerations, researchers can generate reliable and interpretable data from multilabel studies involving MYO7B antibodies, allowing for robust comparative analyses across different experimental conditions.