MYO18A Antibody, HRP conjugated

What is MYO18A and what cellular functions does it perform?

MYO18A is an unconventional myosin that plays several critical roles in cellular function. It is primarily involved in the stabilization and organization of the actin cytoskeleton . More specifically, MYO18A links Golgi membranes to the cytoskeleton and participates in the tensile force required for vesicle budding from the Golgi. This function helps maintain the characteristic flattened shape of the Golgi apparatus and facilitates Golgi membrane trafficking .

Additionally, MYO18A works in concert with LURAP1 and CDC42BPA/CDC42BPB to modulate lamellar actomyosin retrograde flow, which is crucial for cell protrusion and migration. In the immune system, it regulates trafficking, expression, and activation of innate immune receptors on macrophages and helps suppress inflammatory responsiveness through modulation of CD14 trafficking . Recent research has also identified a striated muscle-specific isoform (Myo18Aγ) that appears to complement the functions of conventional class 2 myosins in sarcomeres .

What are the known isoforms of MYO18A and how do they differ in expression patterns?

MYO18A exists in multiple isoforms with distinct tissue distribution and functions:

The expression pattern differs significantly between isoforms. Myo18Aβ is primarily expressed in immature macrophage-like cells, while Myo18Aα expression emerges in mature cells, suggesting different roles in macrophage function . Notably, Myo18Aγ is exclusively expressed in cardiac and skeletal muscle tissues and was only recently identified through specialized detection methods .

What applications can MYO18A antibodies be used for, and what are the recommended dilutions?

MYO18A antibodies have been validated for multiple research applications with specific recommended dilutions:

For HRP-conjugated MYO18A antibodies specifically, the dilutions may need to be optimized based on the conjugation ratio and detection system. It is recommended that researchers titrate the reagent in each testing system to obtain optimal results, as sensitivity may vary between different applications and samples .

How should MYO18A antibodies be stored and handled to maintain reactivity?

For optimal performance of MYO18A antibodies including HRP-conjugated versions, proper storage and handling are essential. The antibodies should be stored at -20°C where they remain stable for one year after shipment . The storage buffer typically consists of PBS with 0.02% sodium azide and 50% glycerol at pH 7.3 .

What is the optimal protocol for using HRP-conjugated MYO18A antibodies in Western blot applications?

For Western blot applications using HRP-conjugated MYO18A antibodies, the following detailed protocol is recommended:

• Sample Preparation:

  • Prepare total protein lysates from tissues or cultured cells (e.g., Jurkat, HeLa, K-562, Neuro-2a, or NIH/3T3 cells, which show positive detection) .

  • Use RIPA buffer with protease inhibitors for extraction.

  • For detecting MYO18A isoforms specifically: use tissue-specific extraction methods, as Myo18Aγ in cardiac tissue requires specialized extraction conditions .

• Gel Electrophoresis:

  • Load 20-50μg of protein per lane.

  • Use low percentage gels (6-8% SDS-PAGE) due to the high molecular weight of MYO18A (calculated 233 kDa; observed 230 kDa or 190 kDa depending on isoform) .

  • Include molecular weight markers that span up to 250 kDa.

• Transfer:

  • Perform wet transfer to PVDF membrane at low voltage (30V) overnight at 4°C to ensure complete transfer of high molecular weight proteins.

• Blocking and Antibody Incubation:

  • Block membrane with 5% non-fat milk in TBST for 1 hour at room temperature.

  • Dilute HRP-conjugated MYO18A antibody at 1:1000-1:6000 in blocking buffer .

  • Incubate membrane with primary antibody solution overnight at 4°C.

  • Since the antibody is HRP-conjugated, secondary antibody incubation is not required.

• Detection:

  • Wash membrane 3-5 times with TBST, 5 minutes each.

  • Develop signal using ECL substrate.

  • Expected bands: 230 kDa (Myo18Aα), 190 kDa (Myo18Aβ), or larger than 230 kDa (Myo18Aγ in muscle tissue) .

How can researchers distinguish between different MYO18A isoforms in their experiments?

Distinguishing between MYO18A isoforms requires careful experimental design and selection of appropriate antibodies and detection methods:

• Antibody Selection Strategy:

  • For detecting all isoforms: Use antibodies against the conserved coiled-coil domain, such as Anti-Myo18A-CC-domain antibodies .

  • For specific detection of Myo18Aα/β: Use antibodies targeting the C-terminal region shared by these isoforms but absent in Myo18Aγ .

  • For specific detection of Myo18Aα (not Myo18Aβ): Target the N-terminal extension containing KE-rich and PDZ domains encoded by exon 1 .

• Molecular Weight Discrimination:

  • Myo18Aα: Appears at approximately 230 kDa

  • Myo18Aβ: Appears at approximately 190 kDa

  • Myo18Aγ: Appears larger than 230 kDa on Western blots

• Tissue-Specific Analysis:

  • Heart and skeletal muscle: Express predominantly Myo18Aγ, which is not detectable with C-terminal antibodies used for Myo18Aα/β .

  • Macrophages: Differential expression with Myo18Aβ in immature cells and Myo18Aα in mature cells .

  • Other cell types: May express combinations of Myo18Aα and Myo18Aβ .

• Confirmatory Approaches:

  • RT-PCR targeting isoform-specific exons to confirm transcript expression.

  • Expression of tagged constructs (EGFP-tagged mouse Myo18Aα and Myo18Aβ) as positive controls .

  • Isoform-specific knockdown using siRNA targeting unique regions.

What troubleshooting steps should be taken when MYO18A antibody shows weak or no signal in IHC applications?

When encountering weak or absent signals in immunohistochemistry applications with MYO18A antibodies, consider these systematic troubleshooting steps:

• Antigen Retrieval Optimization:

  • Primary recommendation: Use TE buffer pH 9.0 for antigen retrieval.

  • Alternative approach: Try citrate buffer pH 6.0 if the primary method is unsuccessful .

  • Extend antigen retrieval time to 20-30 minutes for formalin-fixed tissues.

• Antibody Dilution Adjustment:

  • Start with the recommended dilution range of 1:500-1:2000 .

  • If signal is weak, use more concentrated antibody (1:250-1:500).

  • Perform a dilution series to determine optimal concentration for your specific tissue.

• Incubation Conditions Modification:

  • Extend primary antibody incubation to overnight at 4°C.

  • For HRP-conjugated antibodies, ensure the activity hasn't been compromised by improper storage.

  • Consider adding a signal amplification step (e.g., tyramide signal amplification).

• Tissue-Specific Considerations:

  • For cardiac or skeletal muscle tissues: Standard antibodies may not detect the Myo18Aγ isoform; use coiled-coil domain-specific antibodies .

  • Ensure tissue fixation was optimal; overfixation can mask epitopes.

• Detection System Verification:

  • For HRP-conjugated antibodies: Verify enzyme activity with a control substrate.

  • Include positive control tissues (mouse heart or skeletal muscle) .

  • Run a parallel IHC with an antibody against a housekeeping protein to confirm the protocol works.

How can MYO18A knockdown/knockout models be effectively generated and validated?

Generation and validation of MYO18A knockdown/knockout models requires careful design due to the complexity of isoforms and the essential nature of the gene:

• Knockdown Approaches:

  • siRNA/shRNA targeting: Design multiple siRNAs targeting different regions of the MYO18A transcript.

  • For isoform-specific knockdown: Target unique exons (e.g., exon 1 for Myo18Aα-specific knockdown).

  • Validation control: Include mouse-specific constructs resistant to human siRNA for rescue experiments .

• Knockout Strategies:

  • Global knockout: Note that homozygous knockout of Myo18A is lethal, as indicated by the failure to produce homozygous knockout first mice in breeding experiments .

  • Conditional knockout: Use the Cre-loxP system targeting exons 8-10 as demonstrated in published research .

  • Reporter knockout: Utilize knockout first reporter systems (e.g., lacZ) to simultaneously track expression patterns .

• Validation Methods:

  • Genotyping: PCR-based verification with appropriate primers for detecting wildtype (298 bp product) versus knockout alleles .

  • Protein expression: Western blot with appropriate antibodies depending on which isoforms you're targeting.

  • Functional assays: For Myo18Aα/β, examine Golgi morphology; for Myo18Aγ, assess cardiac and skeletal muscle function .

• Experimental Approaches from Literature:

  • Southern blot analysis to confirm correct targeting of the Myo18a gene .

  • Flp-FRT recombination for deletion of FRT-flanked sequences .

  • PGK-Cre-mediated deletion of loxP-flanked exons .

What is the relationship between MYO18A and the Golgi apparatus, and how can this be studied experimentally?

MYO18A plays a crucial role in Golgi apparatus structure and function, which can be investigated through several experimental approaches:

• Established Golgi-MYO18A Relationship:

  • MYO18A links Golgi membranes to the cytoskeleton via interaction with GOLPH3 (Golgi phosphoprotein 3) .

  • It provides tensile force required for vesicle budding from the Golgi .

  • This interaction helps maintain the characteristic flattened morphology of the Golgi apparatus .

• Experimental Approaches to Study This Relationship:

  • Colocalization Studies:

    • Immunofluorescence using anti-MYO18A antibodies (including HRP-conjugated for EM studies) along with Golgi markers.

    • Live-cell imaging using fluorescently tagged MYO18A constructs to visualize dynamics.

  • Protein-Protein Interaction Analysis:

    • Co-immunoprecipitation to confirm MYO18A-GOLPH3 interactions .

    • Mass spectrometry to identify additional binding partners within the Golgi complex.

  • Functional Studies:

    • siRNA-mediated knockdown of MYO18A to observe effects on Golgi morphology .

    • Rescue experiments using wildtype versus motor-mutant (ATPase-deficient) MYO18A constructs .

    • Vesicle budding assays to quantify the impact of MYO18A depletion.

• Quantitative Analysis Methods:

  • Measure Golgi apparatus size, distribution, and compactness before and after MYO18A manipulation.

  • Track vesicle formation rates from the Golgi using live-cell imaging.

  • Assess Golgi ultrastructure using electron microscopy with immunogold-labeled MYO18A antibodies.

How should researchers design experiments to investigate the role of MYO18A in cardiac and skeletal muscle function?

The discovery of the muscle-specific Myo18Aγ isoform opens new research avenues requiring specialized experimental approaches:

• Isoform-Specific Detection Strategy:

  • Use anti-Myo18A coiled-coil domain antibodies that can detect Myo18Aγ in muscle tissues .

  • Note that antibodies targeting the C-terminal regions of Myo18Aα/β will fail to detect Myo18Aγ due to its unique C-terminus .

  • For transcript analysis, design primers spanning the novel exons identified in Myo18Aγ (GenBank accession number MK268687) .

• Tissue Preparation and Analysis Methods:

  • For protein extraction: Use specialized buffers optimized for myofibrillar proteins.

  • For immunohistochemistry: Employ the recommended antigen retrieval with TE buffer pH 9.0 .

  • For subcellular localization: Combine with sarcomeric markers (e.g., α-actinin, myosin heavy chain).

• Functional Studies Design:

  • In vivo approaches:

    • Conditional knockout: Create muscle-specific Myo18A knockout using muscle-specific Cre drivers (e.g., MCK-Cre).

    • Physiological assessment: Echocardiography for cardiac function, grip strength and running tests for skeletal muscle.

  • In vitro approaches:

    • Primary cardiomyocyte or myoblast cultures with siRNA knockdown.

    • Force generation measurements in isolated muscle fibers.

    • Sarcomere assembly and dynamics using live-cell imaging.

• Molecular Function Investigation:

  • Assess if Myo18Aγ affects conventional class 2 myosins through co-sedimentation or in vitro motility assays .

  • Investigate potential interactions with other sarcomeric proteins through proximity labeling approaches.

  • Examine the effects of Myo18Aγ on actomyosin ATPase activity and actin filament organization.

What considerations are important when analyzing contradictory results between different anti-MYO18A antibodies?

When researchers encounter contradictory results using different anti-MYO18A antibodies, systematic analysis is required:

• Epitope Specificity Considerations:

  • Isoform specificity: Different antibodies target different domains; some fail to detect certain isoforms.

    • Example: Antibodies against C-terminal regions of Myo18Aα/β do not detect Myo18Aγ in cardiac tissue .

    • Recommendation: Use epitope mapping to determine exact binding regions of each antibody.

  • Cross-reactivity assessment:

    • Validate specificity using overexpressed tagged constructs as positive controls.

    • Perform knockdown/knockout validation to confirm specificity.

    • Compare reactivity across human, mouse, and rat samples, as recommended by manufacturers .

• Technical Variable Analysis:

  • Application-specific differences:

    • An antibody may work in WB but not in IHC due to epitope accessibility.

    • Different fixation methods can dramatically affect epitope recognition.

    • Recommended dilutions vary significantly between applications (1:1000-1:6000 for WB vs. 1:500-1:2000 for IHC) .

  • Protocol optimization:

    • For contradictory results in IHC, try both recommended antigen retrieval methods (TE buffer pH 9.0 and citrate buffer pH 6.0) .

    • For Western blot, test different extraction methods, as some may better preserve certain epitopes.

• Resolution Strategy:

  • Use multiple antibodies targeting different regions in parallel.

  • Combine antibody-based detection with orthogonal methods (mass spectrometry, RNA-seq).

  • When publishing, clearly report which antibody was used, its epitope, catalog number, and validation methods.

How can MYO18A antibodies be used to investigate the role of this protein in immune cell function?

MYO18A plays important roles in immune cells, particularly in macrophages, and can be investigated using specialized approaches:

• Isoform Dynamics in Immune Cells:

  • Myo18Aβ is predominantly expressed in immature macrophage-like cells.

  • Myo18Aα expression emerges in mature macrophages, suggesting developmental regulation .

  • Experimental approach: Track isoform switching during macrophage differentiation using isoform-specific antibodies.

• Functional Roles in Macrophages:

  • MYO18A regulates trafficking, expression, and activation of innate immune receptors.

  • It suppresses inflammatory responsiveness via modulation of CD14 trafficking .

  • Experimental design: Use HRP-conjugated MYO18A antibodies for temporal tracking of MYO18A redistribution during immune cell activation.

• Methodological Approaches:

  • Flow cytometry: Use HRP-conjugated or fluorescently labeled MYO18A antibodies to correlate MYO18A expression with macrophage maturation markers.

  • Immunofluorescence microscopy: Track colocalization of MYO18A with CD14 and other immune receptors during activation.

  • Functional assays: Measure cytokine production, phagocytosis, and migration in MYO18A-depleted macrophages.

• Technical Considerations:

  • For flow cytometry applications, permeabilization is required as MYO18A is primarily intracellular.

  • When studying dynamic changes in localization, live-cell imaging with minimal fixation artifacts is recommended.

  • For quantitative studies, HRP-conjugated antibodies can be used for sensitive ELISA-based quantification of MYO18A levels.

What methods can be used to study the interaction between MYO18A and the actin cytoskeleton?

The interaction between MYO18A and actin is central to its cellular functions and can be investigated through multiple complementary approaches:

• Biochemical Interaction Assays:

  • Actin co-sedimentation assays: Mix purified MYO18A with F-actin and ultracentrifuge to determine binding affinity.

  • Actin gliding assays: Assess how MYO18A affects actin filament mobility, noting that MYO18A inhibits the translocation of actin filaments by class 2 myosin .

  • ATPase activity measurements: Determine if actin binding affects MYO18A's ATPase activity.

• Cellular Localization Studies:

  • Co-localization analysis: Use HRP-conjugated MYO18A antibodies alongside fluorescent actin markers.

  • Isoform-specific differences: Myo18Aα (but not Myo18Aβ) localizes to actin filaments and the plasma membrane .

  • Live-cell imaging: Track dynamics of fluorescently tagged MYO18A constructs relative to actin.

• Functional Perturbation Approaches:

  • Domain mapping: Express constructs with mutations in actin-binding domains to determine critical regions.

  • Cytoskeletal disruption: Treat cells with actin-disrupting drugs and observe effects on MYO18A localization.

  • Force measurements: Use traction force microscopy to assess how MYO18A depletion affects cellular force generation.

• Advanced Imaging Techniques:

  • Super-resolution microscopy: Visualize nanoscale organization of MYO18A on actin filaments.

  • FRET analysis: Measure direct interactions between labeled MYO18A and actin in living cells.

  • Correlative light-electron microscopy: Combine immunofluorescence with electron microscopy using HRP-conjugated antibodies for DAB precipitation.

What considerations are important when designing MYO18A detection strategies for specific subcellular compartments?

When targeting MYO18A in specific subcellular locations, researchers should consider these critical factors:

What emerging techniques might improve detection and functional analysis of MYO18A in research settings?

Several cutting-edge techniques show promise for advancing MYO18A research:

• Advanced Imaging Technologies:

  • Cryo-electron tomography: For visualization of MYO18A within native cellular complexes at molecular resolution.

  • Expansion microscopy: To physically enlarge samples for improved visualization of MYO18A in complex structures like sarcomeres.

  • Light-sheet microscopy: For rapid 3D imaging of MYO18A dynamics with reduced phototoxicity in living samples.

• Gene Editing Approaches:

  • CRISPR-Cas9 knock-in strategies: For endogenous tagging of MYO18A with fluorescent proteins or epitope tags without overexpression artifacts.

  • Base editing and prime editing: For introducing specific mutations to study structure-function relationships with minimal off-target effects.

  • Inducible degradation systems: For acute temporal control of MYO18A protein levels.

• Proteomics Innovations:

  • Proximity labeling techniques (BioID, APEX): To identify compartment-specific interaction partners of different MYO18A isoforms.

  • Cross-linking mass spectrometry: For detailed mapping of protein-protein interaction interfaces.

  • Single-cell proteomics: To examine cell-to-cell variation in MYO18A expression and modification state.

• Functional Analysis Methods:

  • Optogenetic control: For spatiotemporal manipulation of MYO18A activity in specific cellular regions.

  • Traction force microscopy: To quantify how MYO18A contributes to cellular force generation and mechanosensing.

  • Organoid models: For studying MYO18A function in more physiologically relevant 3D tissue contexts.

These emerging technologies will likely provide deeper insights into MYO18A's diverse cellular functions and tissue-specific roles, particularly in cardiac and immune cell biology where specialized isoforms play critical roles.

How should researchers interpret MYO18A data in the context of disease models and potential therapeutic applications?

When analyzing MYO18A in disease contexts, researchers should consider these interpretive frameworks:

• Disease Relevance Assessment:

  • Cardiac disorders: Given the essential role of Myo18Aγ in sarcomeric function, analyze potential contributions to cardiomyopathies .

  • Immune dysregulation: Examine MYO18A alterations in inflammatory conditions, considering its role in suppressing inflammatory responses in macrophages .

  • Cell migration disorders: Investigate connections to cancer metastasis based on MYO18A's role in lamellar actomyosin retrograde flow and cell migration .

• Methodological Considerations for Disease Models:

  • Animal models: Consider tissue-specific conditional knockouts rather than global deletion, which is lethal .

  • Patient samples: Use isoform-specific antibodies, as disease may affect specific isoforms differently.

  • Cellular models: Account for cell type-specific expression patterns when designing in vitro disease models.

• Therapeutic Target Assessment Framework:

  • Isoform selectivity: Evaluate whether targeting specific isoforms (e.g., Myo18Aγ for cardiac conditions) could provide therapeutic specificity.

  • Functional domains: Consider targeting specific protein-protein interactions rather than the entire protein.

  • Expression modulation: Assess whether altering MYO18A levels could normalize disease phenotypes in relevant models.

• Translational Research Considerations:

  • Biomarker potential: Evaluate MYO18A as a potential diagnostic or prognostic marker in diseases affecting tissues where it plays critical roles.

  • Target validation: Use multiple independent approaches (genetic, pharmacological) to confirm disease relevance.

  • Model systems: Progress from simple cellular systems to more complex models (organoids, animal models) before clinical translation.