MYO18B Antibody, Biotin conjugated

What is MYO18B and why is it important in research?

MYO18B is an unconventional myosin heavy chain protein primarily expressed in skeletal and cardiac muscles, with lower expression levels in testis. Unlike conventional class II sarcomeric myosins, MYO18B belongs to the class XVIII myosins and contains unique large amino acid domains at both N and C termini that are specific to vertebrates . Recent research has revealed that MYO18B functions as an actin crosslinker that promotes lysosomal exocytosis by facilitating focal adhesion maturation . It plays dual roles depending on its cellular localization: in the cytoplasm, it may be involved in intracellular trafficking within muscle cells, while in the nucleus, it may regulate muscle-specific genes . MYO18B is also implicated in the control of tumor development and progression, as restored MYO18B expression in lung cancer cells has been shown to suppress anchorage-independent growth .

How does a biotin-conjugated MYO18B antibody differ from unconjugated versions?

Biotin-conjugated MYO18B antibodies contain biotin molecules covalently attached to the antibody structure, creating a versatile tool that leverages the strong avidin-biotin interaction system. Unlike unconjugated antibodies (which require secondary detection methods), biotin-conjugated antibodies can be directly detected using streptavidin or avidin conjugated to various reporter molecules (fluorophores, enzymes, or gold particles). This conjugation offers several methodological advantages: (1) enhanced sensitivity due to signal amplification capabilities, (2) flexibility in detection strategies, (3) compatibility with multiplexing experiments, and (4) reduced background in tissues with high endogenous immunoglobulin levels. When selecting between conjugated and unconjugated formats, researchers should consider their specific experimental requirements, including detection systems available, sensitivity needs, and potential for endogenous biotin interference.

What tissues and cell types express MYO18B that can be detected with this antibody?

MYO18B is primarily expressed in striated muscles (both cardiac and skeletal) and at lower levels in testis . Immunolocalization studies have demonstrated that MYO18B protein can be detected in undifferentiated myoblasts (cytoplasmic localization), differentiated myotubes (partial nuclear accumulation), primary cardiomyocytes, and adult muscle sections . In cardiomyocytes, MYO18B sometimes displays a partial sarcomeric pattern alternating with that of alpha-actinin-2 . The punctate cytoplasmic staining pattern is observed in both cardiac and skeletal muscle fibers . Commercial antibodies have been validated for detection of MYO18B in human and mouse samples, with positive immunohistochemistry results specifically noted in mouse heart tissue .

How does MYO18B's role in lysosomal exocytosis impact experimental design for studying cancer cell behavior?

Recent research has uncovered that MYO18B positively regulates lysosomal exocytosis by promoting focal adhesion maturation . This function has significant implications for cancer research, particularly regarding paucimannose glycans, which are truncated N-glycan structures that are increased in many cancer cells. These glycans are produced by lysosomal glycosidases and secreted via lysosomal exocytosis, which preferentially occurs near focal adhesions .

When designing experiments to study cancer cell behavior in relation to MYO18B, researchers should consider:

• Focal adhesion analysis: Include techniques to visualize and quantify focal adhesion formation and maturation (e.g., immunofluorescence for focal adhesion proteins like paxillin, vinculin)

• Mechanosensing pathways: Incorporate analyses of mechanosensitive channels like PIEZO1, which locally activates at focal adhesions to import Ca²⁺ necessary for lysosome-plasma membrane fusion

• Glycomic profiling: Include methods to detect and quantify paucimannose glycans on the cell surface and in secreted material

• Spatial resolution techniques: Employ high-resolution microscopy to visualize the spatial relationship between lysosomes, focal adhesions, and the plasma membrane

When using biotin-conjugated MYO18B antibodies in this context, researchers should establish proper controls to distinguish between MYO18B-specific effects and general changes in the actin cytoskeleton or focal adhesion dynamics.

What are the critical considerations when using biotin-conjugated MYO18B antibodies in tissues with high endogenous biotin levels?

Tissues with high endogenous biotin levels (particularly liver, kidney, brain, and adipose tissue) present specific challenges when using biotin-conjugated antibodies. For research involving MYO18B in these tissues, consider the following methodological approaches:

• Endogenous biotin blocking: Implement a pre-blocking step using unconjugated streptavidin or avidin (0.1-1 mg/mL) for 15-30 minutes prior to primary antibody incubation, followed by thorough washing.

• Alternative detection strategies: When studying MYO18B in high-biotin tissues, consider:

  • Using unconjugated primary antibodies with non-biotin detection systems

  • Employing directly-labeled primary antibodies

  • Utilizing tyramide signal amplification methods for enhanced sensitivity without biotin

• Validation controls:

  • Include a streptavidin-only control (no primary antibody) to assess endogenous biotin levels

  • Compare staining patterns with alternative MYO18B antibody formats

  • Use tissues known to be negative for MYO18B expression as specificity controls

• Signal quantification considerations: When analyzing images from high-biotin tissues, implement computational approaches to subtract background signal and normalize to control regions.

How might MYO18B mutations affect antibody binding and experimental interpretation?

Mutations in MYO18B have been implicated in severe nemaline myopathy and cardiomyopathy . When studying patient samples or experimental models with MYO18B mutations, researchers must consider several factors that could affect antibody binding and data interpretation:

• Epitope accessibility: Mutations may alter protein folding, affecting antibody epitope exposure. This is particularly relevant for antibodies targeting regions near mutation hotspots.

• Truncated protein detection: Nonsense mutations, like the homozygous stop codon described in a severe nemaline myopathy case , result in truncated proteins. Antibody selection should consider the location of the targeted epitope relative to the truncation:

  • N-terminal antibodies may detect truncated proteins (as demonstrated in a patient study showing positive N-terminal antibody staining but negative C-terminal antibody staining)

  • C-terminal antibodies may yield false negatives in samples with C-terminal truncations

• Subcellular localization changes: Mutations may alter MYO18B's normal distribution between cytoplasm and nucleus, affecting staining patterns. Compare subcellular distribution patterns between wild-type and mutant samples using multiple detection methods.

• Expression level variations: Some mutations affect protein stability rather than antibody binding directly. Quantitative approaches like Western blotting should complement immunostaining to assess protein levels accurately.

What are the optimal fixation and antigen retrieval methods for MYO18B immunodetection?

The selection of appropriate fixation and antigen retrieval methods is critical for successful immunodetection of MYO18B. Based on validated protocols:

Fixation Recommendations:

• For cell cultures: 4% paraformaldehyde for 15-20 minutes at room temperature preserves MYO18B structure while maintaining cellular architecture

• For tissue sections: 10% neutral buffered formalin with controlled fixation time (12-24 hours) to prevent overfixation

Antigen Retrieval Protocols:
For formalin-fixed paraffin-embedded tissues, heat-induced epitope retrieval is recommended with the following options:

For optimal results with biotin-conjugated antibodies, an additional blocking step to neutralize endogenous biotin activity is strongly recommended after antigen retrieval but before antibody application.

What protocol modifications are needed when using biotin-conjugated MYO18B antibodies for multiple labeling experiments?

Multiple labeling experiments with biotin-conjugated MYO18B antibodies require careful protocol design to ensure specificity and prevent cross-reactivity. Follow these methodological guidelines:

• Sequential Detection Approach:

  • Complete the biotin-streptavidin detection cycle for MYO18B before introducing additional primary antibodies

  • Block remaining biotin binding sites with excess biotin (1 mg/mL) after MYO18B detection

  • Use directly labeled primary antibodies for subsequent targets to avoid cross-reactivity with biotin-avidin systems

• Antibody Selection Considerations:

  • Choose primary antibodies from different host species to minimize cross-reactivity

  • For co-localization with other muscle proteins (e.g., alpha-actinin-2, which shows alternating patterns with MYO18B in cardiomyocytes) , select antibodies validated for multiple labeling

• Optimized Multiplexing Protocol:

  • Perform antigen retrieval

  • Block endogenous biotin

  • Apply biotin-conjugated MYO18B antibody at optimal dilution

  • Detect with fluorophore-conjugated streptavidin

  • Block remaining biotin/streptavidin binding sites

  • Apply subsequent primary antibodies

  • Detect with non-biotin secondary detection systems

  • Include single-staining controls for each antibody

• Spectral Considerations:

  • Select fluorophores with minimal spectral overlap

  • Include single-color controls for computational spectral unmixing if needed

What are the recommended dilutions and incubation conditions for optimal detection of MYO18B?

Based on validated protocols for commercial MYO18B antibodies, the following dilutions and conditions are recommended for optimal detection:

For biotin-conjugated antibodies specifically, these dilutions may need adjustment based on the degree of biotin conjugation and the detection system employed. It is strongly recommended to perform titration experiments to determine optimal conditions for each specific lot and application.

How can researchers troubleshoot weak or absent MYO18B signal in muscle tissue samples?

When experiencing weak or absent MYO18B signal in muscle tissue samples despite using validated biotin-conjugated antibodies, consider these methodological troubleshooting approaches:

• Tissue-specific expression considerations:

  • Verify correct tissue type: MYO18B is primarily expressed in striated muscles (cardiac and skeletal) and testis

  • Consider developmental stage: MYO18B expression increases after myoblast differentiation into myotubes

• Technical optimization steps:

  • Antigen retrieval: Try alternative methods, particularly TE buffer (pH 9.0) which has been specifically validated for MYO18B detection

  • Signal amplification: Implement tyramide signal amplification to enhance detection sensitivity

  • Antibody concentration: Titrate to higher concentrations (1:50-1:250) for immunofluorescence applications

  • Incubation time: Extend primary antibody incubation to overnight at 4°C

• Antibody epitope considerations:

  • Antibody target region: If studying potential truncated MYO18B variants, verify whether your antibody targets N-terminal or C-terminal regions

  • Multiple antibody approach: Use antibodies targeting different epitopes to confirm results, particularly in cases of suspected mutations

• Control implementations:

  • Positive control: Include tissues with known MYO18B expression (e.g., mouse heart tissue)

  • Subcellular localization assessment: Check for both nuclear and cytoplasmic staining patterns, as MYO18B distributes to both compartments

What strategies can address cross-reactivity issues when using biotin-conjugated MYO18B antibodies?

Cross-reactivity can compromise experimental results when using biotin-conjugated antibodies. Implement these methodological approaches to address and minimize such issues:

• Antibody validation strategies:

  • Epitope mapping: Verify the specific epitope recognized by your antibody to assess potential cross-reactivity

  • Pre-absorption controls: Pre-incubate antibody with purified MYO18B protein before staining to confirm specificity

  • Genetic controls: When possible, use MYO18B-knockout or knockdown samples as negative controls

• Protocol modifications to reduce non-specific binding:

  • Increase blocking stringency: Use 5-10% serum from the same species as secondary reagents plus 1-3% BSA

  • Add protein competitors: Include 0.1-0.3% Triton X-100, 0.05-0.1% Tween-20, and 0.1-0.3 M NaCl in antibody diluents

  • Reduce antibody concentration: Titrate to determine minimal effective concentration

  • Implement avidin/biotin blocking: For tissues with high endogenous biotin

• Detection system considerations:

  • Use highly cross-adsorbed streptavidin conjugates

  • Consider direct detection methods instead of amplification systems when cross-reactivity persists

• Data validation approaches:

  • Compare staining patterns with unconjugated MYO18B antibodies

  • Confirm key findings with orthogonal methods (e.g., RNA expression, mass spectrometry)

How should researchers interpret changes in MYO18B subcellular localization across different experimental conditions?

MYO18B exhibits dynamic subcellular localization between cytoplasm and nucleus depending on cell type and differentiation state. When analyzing subcellular distribution patterns:

• Baseline localization patterns to recognize:

  • Undifferentiated myoblasts: Primarily cytoplasmic localization

  • Differentiated myotubes: Both cytoplasmic and nuclear localization, with accumulation in a subset of myonuclei

  • Cardiomyocytes: Punctate cytoplasmic pattern, sometimes with partial sarcomeric arrangement alternating with alpha-actinin-2

• Quantitative analysis approaches:

  • Implement nuclear/cytoplasmic fluorescence intensity ratio measurements

  • Use co-localization coefficients with nuclear markers (DAPI) and cytoskeletal markers (alpha-actinin)

  • Employ subcellular fractionation followed by Western blotting to biochemically confirm microscopy observations

• Functional correlation strategies:

  • Correlate MYO18B localization changes with differentiation markers expression

  • Assess relationship between MYO18B nuclear accumulation and muscle-specific gene expression

  • Evaluate connection between cytoplasmic MYO18B patterns and lysosomal exocytosis events

• Experimental perturbations to consider:

  • Test effects of cytoskeletal disrupting agents on MYO18B localization

  • Examine localization changes following mechanical stimulation of cells (given MYO18B's role in mechanosensing pathways)

  • Assess calcium signaling effects on MYO18B distribution (considering its connection to PIEZO1 channel activity)

How does MYO18B's role in lysosomal exocytosis impact our understanding of cancer cell glycosylation patterns?

Recent breakthrough research published in January 2025 revealed that MYO18B plays a previously unrecognized role in regulating lysosomal exocytosis by promoting focal adhesion maturation . This function has significant implications for understanding cancer cell biology, particularly regarding paucimannose glycans.

The study demonstrated that paucimannosidic proteins are produced by lysosomal glycosidases and secreted through lysosomal exocytosis, which preferentially occurs near focal adhesions . MYO18B, functioning as an actin crosslinker, facilitates focal adhesion maturation, thereby enabling this localized exocytosis process .

This mechanistic insight connects cellular mechanosensing with glycosylation patterns through the following pathway:

• MYO18B promotes focal adhesion maturation

• Mechanosensitive PIEZO1 channels activate locally at these adhesion sites

• PIEZO1 mediates Ca²⁺ influx required for lysosome-plasma membrane fusion

• Lysosomal exocytosis releases paucimannosidic proteins to the cell surface

For cancer researchers, these findings suggest that MYO18B expression levels may influence the glycosylation signature of cancer cells, potentially affecting their interactions with the tumor microenvironment. Future therapeutic strategies might target components of this pathway to modulate cancer cell behavior through altered glycosylation patterns.