MYO7A Recombinant Monoclonal Antibody

What is MYO7A and what are its primary functions in cellular processes?

MYO7A is a large, multifunctional motor protein (calculated molecular weight of 254,390 Da) that plays diverse roles in cellular processes. Its primary functions include:

• Maintenance of stereocilia structure and function in inner ear hair cells

• Visual sensory functions in the retina

• Intracellular transport and trafficking

• Maintenance of microvilli and cellular adhesion

• Vesicle trafficking and melanosome transport

• Formation of protein complexes with other sensory proteins

MYO7A is particularly important in auditory and visual pathways, where mutations can lead to disorders affecting hearing, vision, and other cellular processes . The protein forms complexes with partners like PDZD7 (a paralog of USH1C and DFNB31) and is associated with membranes in various cell types .

How are MYO7A recombinant monoclonal antibodies generated?

The production of MYO7A recombinant monoclonal antibodies follows a sophisticated molecular biology process:

• The MYO7A antibody-encoding gene is inserted into expression vectors

• These vectors are delivered into host cells using polyethyleneimine-mediated transfection

• Host cells containing these vectors are cultured to produce and secrete the antibodies

• The antibodies undergo affinity chromatography purification

• Quality control includes ELISA and flow cytometry (FC) tests to confirm target recognition

• The antibodies are validated for specific reactivity to human MYO7A protein

This recombinant approach provides advantages over traditional hybridoma-based monoclonal antibody production, including better batch-to-batch consistency and the ability to engineer specific binding properties.

What applications are MYO7A antibodies validated for in research settings?

MYO7A antibodies have been validated for multiple research applications, with varying recommended dilutions based on the specific assay:

Researchers should note that specific antibodies like the one described in source react with both human and mouse MYO7A, making them versatile for cross-species studies. Each application requires optimization of conditions for specific experimental systems.

How can MYO7A antibodies be utilized to study protein complexes in stereocilia?

MYO7A forms critical complexes with other proteins in stereocilia, making antibody-based approaches valuable for studying these interactions. A methodological approach includes:

• Stabilize protein complexes using chemical crosslinking with membrane-impermeant crosslinker DTSSP, which enhances detection of low-abundance membrane protein interactions

• Use stringent extraction and washing conditions to minimize dissociation of protein complexes that include extracellular domains

• Employ immunoaffinity purification using anti-MYO7A antibodies (such as the 138-1 monoclonal antibody for chicken MYO7A)

• Remove nonspecific proteins by first passing extracts over control beads constructed with purified IgG

• Apply the unbound material to anti-MYO7A antibody-conjugated beads

• After thorough washing, elute with SDS and analyze by shotgun and targeted mass spectrometry

This approach has successfully identified MYO7A complexes with PDZD7, USH1C, and CDH23, suggesting a functional network in stereocilia . The high enrichment (approximately 10,000-fold) of MYO7A through this method allows detection of even rare interacting partners.

What strategies should be employed when validating MYO7A antibody specificity for studies in mutant models?

When validating MYO7A antibody specificity in mutant models, researchers should implement a multi-tiered approach:

• Genetic controls: Use MYO7A-null models (such as shaker-1 Myo7a^-/-^ mice or Myo7a^4626SB/4626SB^ mutants) as negative controls to confirm antibody specificity

• Epitope mapping: Verify antibody recognition using antibodies directed against different portions of MYO7A (N-terminal, middle, and C-terminal regions)

• Cross-reactivity assessment: Test antibody reactivity against related myosin family members

• Tagged protein validation: Compare staining patterns between antibodies recognizing native MYO7A and those recognizing epitope tags (such as HA) in tagged transgene expression systems

• Immunoblotting validation: Confirm the detection of appropriately sized bands in wild-type but not mutant tissues

Research has demonstrated that proper antibody validation is critical when studying MYO7A, as demonstrated in studies using shaker-1 mice where antibodies detecting the middle portion of MYO7A only detected expression when both 5′ and 3′ vectors were used to infect cells .

What considerations are important when designing experiments to study MYO7A-mediated functions in sensory cells?

Designing experiments to study MYO7A-mediated functions requires careful consideration of multiple factors:

• Tissue-specific expression patterns: MYO7A functions differ between auditory hair cells, vestibular hair cells, and retinal cells, necessitating tissue-specific experimental designs

• Subcellular localization: Consider the distribution of MYO7A within different cellular compartments (stereocilia, cell body, synapses)

• Temporal dynamics: Account for developmental timing and age-related changes in MYO7A expression and function

• Genetic background effects: Control for genetic background influences on phenotypic expression in MYO7A mutant models

• Redundancy with other myosins: Consider potential functional overlap with other unconventional myosins

• Protein complex formation: Design experiments that capture MYO7A's participation in different protein complexes

Research has shown that MYO7A is multifunctional, interacting with several different protein complexes in stereocilia and carrying out various transport and functional roles . Therefore, experimental designs must account for these multiple functions rather than assuming a single role.

What technical challenges arise when using MYO7A antibodies for immunoprecipitation of protein complexes?

Immunoprecipitation of MYO7A protein complexes presents several technical challenges:

• Low abundance: MYO7A and its complexes are rare within stereocilia, requiring approximately 10^6-fold enrichment from dissected inner ears for definitive detection

• Complex integrity preservation: Maintaining intact protein complexes during extraction and purification is challenging, often requiring chemical crosslinking

• Nonspecific binding: Extensive washing is required to reduce nonspecific binding, which biases analysis toward high-affinity interactions

• Starting material limitations: Large-scale purification requires substantial starting material (hundreds of ear-equivalents)

• Sequential purification requirements: Multiple purification steps may be needed to achieve sufficient enrichment

Research suggests that future studies should employ a second antibody purification step following initial precipitation with anti-MYO7A antibodies to generate purer complexes. This might involve using antibodies against known interacting partners like anti-PDZD7 .

How can dual-AAV vector-mediated MYO7A expression be optimized in inner ear research?

Optimizing dual-AAV vector-mediated MYO7A expression for inner ear research requires attention to several critical factors:

• Vector design: Use hybrid dual-AAV vectors with recombinogenic sequences (e.g., AP coding sequence) and splice donor/acceptor sites to promote efficient recombination and transcript processing

• Vector serotype selection: AAV8(Y733F) has shown efficacy for inner ear delivery

• Promoter choice: The ubiquitous CMV/chicken β-actin (smCBA) promoter has proven effective for MYO7A expression

• Injection technique: Employ posterior semicircular canal (PSC) injection for efficient cochlear transduction

• Timing of intervention: Administer therapy early enough to prevent irreversible damage

• Vector dose optimization: Titrate vector doses to achieve sufficient expression while minimizing toxicity

Studies have demonstrated that hybrid dual-AAV8(Y733F)-MYO7A vectors can restore MYO7A protein expression in transduced shaker-1 inner hair cells (IHCs) and outer hair cells (OHCs), significantly improving hair cell survival compared to untreated mutants .

What protocols are recommended for validating MYO7A antibodies across different species?

When validating MYO7A antibodies for cross-species reactivity, researchers should implement the following protocols:

• Sequence alignment analysis: Compare MYO7A sequences across target species to identify conserved and variable regions

• Epitope selection: For recombinant antibodies, target highly conserved regions (the AA range 830-910 has been used successfully for antibodies reacting with both human and mouse MYO7A)

• Multiple application testing: Validate antibodies in different applications (WB, ELISA, FC, IHC) for each species

• Positive control samples: Use well-characterized positive control samples from each species

• Negative control samples: Include MYO7A-null or knockdown samples when available

• Cross-reactivity panel: Test against closely related myosin family members

Researchers should be aware that antibody validation is application-specific – an antibody that works well for Western blotting may not perform adequately in immunohistochemistry or flow cytometry applications.

How should researchers interpret data from MYO7A localization studies in different sensory cell types?

Interpreting MYO7A localization data across different sensory cell types requires careful consideration of:

• Cell type-specific distribution patterns: MYO7A localizes differently in various cell types:

  • In hair cells: Concentrated in stereocilia, cuticular plate, and cell body

  • In retinal cells: Associated with connecting cilium and melanosomes

• Fixation and permeabilization effects: Different protocols can affect epitope accessibility and apparent distribution patterns

• Antibody specificity considerations: Different antibodies targeting different regions of MYO7A (N-terminal, middle, or C-terminal portions) may show slightly different localization patterns

• Co-localization analysis: Examine co-distribution with known interacting partners (PDZD7, USH1C, CDH23) to validate functional associations

• Resolution limitations: Consider the diffraction limit of conventional microscopy versus super-resolution techniques when interpreting apparent co-localization

Research has demonstrated that MYO7A's localization correlates with its diverse functions, including interactions with stereocilia membranes and protein complexes at tip links and ankle links .

What are the key considerations when analyzing MYO7A complex formation in mass spectrometry data?

When analyzing mass spectrometry data for MYO7A complexes, researchers should consider:

• Enrichment analysis: Compare relative abundance of proteins in immunoprecipitates versus starting material to calculate immunoaffinity enrichment ratios (MYO7A itself should show high enrichment, ~10-fold or greater)

• Specificity controls: Distinguish proteins detected only in MYO7A immunoprecipitates from those in control samples

• Known complex components: Verify presence of established MYO7A-interacting proteins (USH1C, USH1G, CDH23) as internal validation

• Abundance estimates: Consider that even after 10,000-fold purification, MYO7A complex components may be present at low levels

• Crosslinking effects: Account for potential artificial associations introduced by chemical crosslinking

• Stoichiometry limitations: Recognize that additional purification steps are required for accurate stoichiometric determination of complex components

Research has shown that despite challenges, large-scale purification of protein complexes from stereocilia can identify interacting components that may not be detected by other strategies .

How might MYO7A antibodies be utilized in therapeutic development for Usher syndrome?

MYO7A antibodies have significant potential in therapeutic development for Usher syndrome through multiple approaches:

• Biomarker development: Use antibodies to measure MYO7A expression levels and localization as biomarkers for therapeutic efficacy

• Vector development assessment: Employ antibodies to evaluate dual-AAV vector-mediated MYO7A expression in preclinical models

• Gene therapy monitoring: Use antibodies to assess restoration of MYO7A expression following gene therapy in shaker-1 mice or human tissues

• Drug screening: Apply antibodies in high-throughput screens for compounds that stabilize mutant MYO7A or enhance its function

• Therapeutic antibody engineering: Develop antibody-based therapeutics that might stabilize MYO7A complexes or enhance their function

Research has demonstrated that dual-AAV vector delivery of human MYO7A cDNA can restore MYO7A expression in mouse models, with significant preservation of inner hair cells compared to untreated mutants , suggesting promising therapeutic avenues.

What roles might MYO7A play in protein complexes beyond the currently established interactions?

Current research suggests MYO7A may participate in additional protein complexes and functions beyond those currently established:

• Novel binding partners: Further immunoprecipitation and mass spectrometry studies may reveal currently unknown interaction partners

• Trafficking complexes: MYO7A likely participates in various cargo trafficking complexes beyond those characterized in stereocilia

• Transcriptional regulation: Potential nuclear functions have not been fully explored

• Signaling pathway integration: MYO7A may link mechanical stimuli to intracellular signaling cascades

• Immune system roles: Possible functions in specialized immune cells have not been extensively investigated

Research has indicated that MYO7A is a multifunctional protein interacting with several different protein complexes in stereocilia and carrying out transport and functional roles , suggesting many aspects of its biology remain to be discovered.

What are the optimal protocols for using MYO7A antibodies in immunofluorescence studies of inner ear tissues?

Immunofluorescence studies of MYO7A in inner ear tissues require specialized protocols:

• Tissue preparation:

  • Fresh dissection or rapid fixation is critical

  • Fixation with 4% paraformaldehyde for 30-60 minutes preserves structure while maintaining epitope accessibility

  • Consider specialized fixatives for transmission electron microscopy studies

• Decalcification considerations:

  • For intact cochlea, use EDTA-based decalcification to preserve antigenicity

  • Monitor decalcification progress to avoid over-processing

• Permeabilization optimization:

  • Test different detergents (Triton X-100, saponin) at various concentrations

  • Balance permeabilization for antibody access while preserving delicate stereocilia structures

• Antibody incubation:

  • Extended incubation times (overnight at 4°C) improve penetration

  • Use antibodies directed against different portions of MYO7A (N-terminal, middle, and C-terminal) to validate findings

• Controls:

  • Include MYO7A-deficient tissues (e.g., from shaker-1 mice) as negative controls

  • Use dual-labeling with established hair cell markers for reference

These approaches have successfully demonstrated MYO7A expression patterns in both normal and genetically modified inner ear tissues .

How can researchers quantitatively assess MYO7A expression levels in different experimental conditions?

Quantitative assessment of MYO7A expression requires rigorous methodological approaches:

• Western blot quantification:

  • Use purified recombinant MYO7A standards for absolute quantification

  • Normalize to appropriate housekeeping proteins or total protein

  • Employ fluorescent secondary antibodies for improved linear range

• ELISA-based quantification:

  • Develop sandwich ELISA using different epitope-targeting antibodies

  • Generate standard curves with recombinant MYO7A

• Flow cytometry:

  • Optimize cell permeabilization protocols for this large intracellular protein

  • Use recommended antibody dilutions (1:50-1:200) for FC applications

  • Include proper controls for autofluorescence and nonspecific binding

• Image analysis:

  • Apply consistent acquisition parameters across samples

  • Use automated, threshold-based quantification

  • Normalize to cell number or tissue area

  • Account for regional variations in expression (apex vs. middle turn vs. base of cochlea)

Research has demonstrated regional differences in MYO7A expression following treatment, with measurements showing 14 ± 1.2 cells/136 μm at the apex, 14 ± 1.9 cells/136 μm at the middle turn, and 7.3 ± 1.8 cells/136 μm at the base of the cochlea .