MYO5A Antibody

What is MYO5A and what are its key biological functions?

MYO5A (Myosin VA) is a processive actin-based motor protein with a molecular weight of approximately 215 kDa that moves in large steps approximating the 36-nm pseudo-repeat of the actin filament . This protein plays critical roles in multiple cellular processes including melanosome transport, vesicle transport to the plasma membrane, and polarization processes involved in dendrite formation . In neuroplasticity contexts, MYO5A interacts with proteins that influence synaptic plasticity and vesicle trafficking, such as synaptophysin and synapsin, supporting efficient synaptic signaling . The protein is also known by several alternative names including d-120J, Dbv, MVa, MYO5, myosin V, and GS1 .

What species reactivity can be expected with MYO5A antibodies?

Most commercially available MYO5A antibodies demonstrate validated reactivity with human, mouse, and rat samples . Some antibodies have predicted or potential reactivity with additional species such as pig, bovine, horse, sheep, rabbit, dog, and chicken, though these require experimental validation before use in critical research applications . When selecting an antibody, researchers should prioritize those with demonstrated reactivity to their species of interest and validate reactivity independently in their experimental systems.

What are the standard applications for MYO5A antibodies?

MYO5A antibodies are validated for multiple laboratory techniques including:

The observed molecular weight for MYO5A in Western blot applications is typically 210-220 kDa . Application-specific optimization is essential for achieving optimal results with any antibody.

What are the optimal protocols for MYO5A detection in Western blot?

For Western blot detection of MYO5A, researchers should consider the following protocol parameters:

• Sample preparation: Fresh tissue lysates from brain tissue provide reliable positive controls

• Protein separation: Standard SDS-PAGE with particular attention to effective separation of high molecular weight proteins

• Expected band size: 210-220 kDa

• Recommended dilution: 1:500-1:2000 for most antibodies

• Incubation conditions: Room temperature for 1.5 hours has been validated in published protocols

• Detection system: Standard secondary antibody and chemiluminescence detection systems are compatible

Given the high molecular weight of MYO5A, extended transfer times or specialized transfer methods for large proteins may be necessary to ensure complete transfer to the membrane.

What antigen retrieval methods are recommended for MYO5A immunohistochemistry?

For optimal MYO5A detection in paraffin-embedded tissue sections, heat-mediated antigen retrieval with Tris-EDTA buffer at pH 9.0 has been experimentally validated . This approach has shown strong, specific staining in mouse brain tissue sections . As an alternative, citrate buffer at pH 6.0 can also be used for antigen retrieval, though comparative performance should be assessed empirically . Optimization of antigen retrieval conditions is particularly important for detection of high molecular weight proteins like MYO5A where epitope masking during fixation can significantly impact antibody accessibility.

What are the optimal storage conditions for MYO5A antibodies?

To maintain MYO5A antibody stability and activity:

• Storage temperature: -20°C is recommended for most preparations

• Buffer composition: PBS with 0.02% sodium azide and 50% glycerol at pH 7.3 is typically used

• Aliquoting: Generally considered unnecessary for -20°C storage

• Additional components: Some smaller volume preparations (20μl) may contain 0.1% BSA as a stabilizer

Following these storage recommendations helps preserve antibody activity and specificity over extended periods, though antibody performance should be periodically validated, particularly for critical applications.

How can I validate the specificity of a MYO5A antibody?

Comprehensive validation of MYO5A antibody specificity should include:

• Positive control analysis: Testing with mouse or rat brain tissue, which express MYO5A at high levels

• Expected molecular weight confirmation: Verifying a single band at 210-220 kDa in Western blot applications

• Multi-application validation: Confirming consistent detection patterns across different techniques (WB, IHC, IF)

• Antibody comparison: Testing multiple antibodies targeting different epitopes of MYO5A

• Knockdown controls: When possible, using siRNA or shRNA to reduce MYO5A expression as a negative control

• Peptide competition: Performing blocking experiments with the immunizing peptide

This multi-faceted approach ensures that observed signals truly represent MYO5A rather than non-specific binding or cross-reactivity with related proteins.

What tissue samples are most appropriate for studying MYO5A?

Based on validated experimental data, the following samples are recommended for MYO5A investigations:

• Brain tissue: Consistently validated as a reliable positive control for MYO5A expression

• Neuronal cultures: Appropriate for studying MYO5A's role in dendrite formation and synaptic signaling

• Melanocytes: Suitable for examining MYO5A's function in melanosome transport

• Secretory cells: Valuable for investigating vesicle trafficking mechanisms

The choice of experimental system should align with the specific aspect of MYO5A biology under investigation, with brain tissue serving as a well-validated positive control for most applications.

What controls should be included in MYO5A expression studies?

Rigorous experimental design for MYO5A studies should incorporate:

• Technical controls: Antibody specificity controls (secondary-only, isotype controls)

• Loading controls: Appropriate housekeeping proteins for normalization in quantitative studies

• Biological controls: Tissues known to express or lack MYO5A expression

• Methodological controls: Antibody dilution series to ensure linearity of signal

• Knockout/knockdown controls: When available, to confirm specificity

How can I study MYO5A interactions with synaptic proteins?

To investigate MYO5A's role in synaptic function and neuroplasticity:

• Co-immunoprecipitation: Use MYO5A antibodies to pull down protein complexes, then probe for interacting partners like synaptophysin and synapsin

• Proximity ligation assay: Visualize protein-protein interactions in situ with single-molecule resolution

• Super-resolution microscopy: Examine co-localization of MYO5A with synaptic proteins at the nanoscale

• FRET/FLIM analysis: Assess dynamic protein interactions in live neuronal cultures

• Subcellular fractionation: Isolate synaptic vesicles and analyze MYO5A association

These approaches can reveal how MYO5A contributes to synaptic plasticity and neurotransmitter release through its interactions with other synaptic proteins .

What approaches are effective for studying MYO5A in vesicle trafficking?

To examine MYO5A's function as a motor protein in vesicle transport:

• Live-cell imaging: Track fluorescently-tagged vesicles in the presence or absence of functional MYO5A

• Single-molecule tracking: Analyze movement parameters including velocity, processivity, and step size

• Optogenetic approaches: Control MYO5A activity with light to examine acute effects on vesicle mobility

• Dominant-negative constructs: Express mutant forms of MYO5A to disrupt specific functions

• Correlative light-electron microscopy: Connect molecular-level MYO5A localization with ultrastructural context

These methodologies can reveal how MYO5A contributes to the directed transport of vesicles to the plasma membrane and other cellular destinations.

How can I distinguish between MYO5A and other myosin V family members?

Strategies to differentiate MYO5A from related proteins (MYO5B, MYO5C):

• Epitope selection: Use antibodies targeting unique regions with minimal sequence homology

• Isoform-specific knockdown: Selectively reduce expression of individual myosin V proteins

• Expression pattern analysis: Examine tissue-specific distribution patterns (MYO5A is enriched in brain and melanocytes)

• Functional complementation: Test whether one myosin V protein can rescue phenotypes caused by loss of another

• Cargo specificity: Identify vesicle populations uniquely transported by each myosin V protein

These approaches are critical for attributing specific cellular functions to MYO5A rather than related myosin motor proteins.

Why might Western blot detection of MYO5A fail despite proper technique?

Several factors could contribute to unsuccessful MYO5A detection:

The high molecular weight of MYO5A (210-220 kDa) presents particular challenges for protein transfer and detection that may require specialized approaches .

How should multiple bands in MYO5A Western blots be interpreted?

When multiple bands appear in Western blot analysis:

• Full-length MYO5A should appear at 210-220 kDa

• Lower molecular weight bands may represent:

  • Proteolytic degradation products

  • Alternatively spliced isoforms

  • Post-translationally modified forms

  • Cross-reactivity with related myosin family members

• Higher molecular weight bands could indicate:

  • Protein aggregation

  • Post-translational modifications like ubiquitination

  • Incomplete denaturation

Comparison with published literature and additional validation experiments (such as mass spectrometry analysis) can help identify the nature of unexpected bands.

What are common pitfalls in immunofluorescence detection of MYO5A?

Challenges in IF/ICC applications include:

• Background fluorescence: Optimize blocking conditions, use purified antibody preparations

• Weak signal: Ensure adequate permeabilization for access to intracellular MYO5A

• Non-specific binding: Include appropriate negative controls (secondary antibody only, isotype controls)

• Variable expression: Confirm cell-type specific expression patterns in heterogeneous cultures

• Epitope masking: Different fixation methods may affect epitope accessibility

The large size of MYO5A may require optimized permeabilization protocols to ensure antibody accessibility to all cellular compartments.

What factors should guide MYO5A antibody selection?

Key considerations for choosing an appropriate MYO5A antibody:

The ideal antibody selection balances these factors based on the specific requirements of the planned experiments.

How do antibodies against different MYO5A epitopes compare?

Performance comparison of antibodies targeting different regions:

• N-terminal antibodies: May detect all isoforms but could be affected by binding partners

• C-terminal antibodies: Such as those targeting amino acids 1350-1550 may miss splice variants lacking this region

• Middle region antibodies: Often provide good specificity but potential for cross-reactivity with homologous regions in other myosin family members

• Functional domain antibodies: Allow targeted study of specific protein domains and their functions

What are the advantages of rabbit polyclonal versus other MYO5A antibodies?

Rabbit polyclonal antibodies, which are commonly available for MYO5A detection , offer several advantages:

• Recognize multiple epitopes, potentially increasing detection sensitivity

• Often work effectively across multiple applications (WB, IHC, IF)

• Generally available at lower cost than monoclonal alternatives

• May maintain reactivity even if some epitopes are masked or modified