MYH15 Antibody

What is MYH15 and where is it expressed in human tissues?

MYH15 (Myosin Heavy Chain 15) is a myosin heavy chain isoform that has been identified in several human tissues. Recent immunohistochemical studies have demonstrated its expression in human muscle spindles, particularly in the extracapsular region of bag fibers . Beyond musculoskeletal tissues, MYH15 is also expressed in the airway epithelium, vascular endothelium, and inflammatory cells . Its expression was previously reported in extraocular muscles, but recent research has expanded our understanding of its tissue distribution . When designing experiments to detect MYH15, researchers should consider this tissue-specific expression pattern to properly interpret results and establish appropriate controls.

How do MYH15 antibodies differentiate between MYH15 and other myosin heavy chain isoforms?

Distinguishing MYH15 from other myosin heavy chain isoforms requires highly specific antibodies due to the structural similarities between myosin family members. Effective MYH15 antibodies are designed to target unique epitopes found in the MYH15 protein that are not present in related myosin isoforms. In immunohistochemical studies, researchers have utilized a specific set of antibodies to demonstrate nine different isoforms (15, slow-tonic, 1, α, 2a, 2x, 2b, embryonic, neonatal) in intrafusal fibers of muscle spindles .

When validating antibody specificity, researchers should:

• Perform Western blot analysis to confirm binding to proteins of the expected molecular weight

• Include appropriate positive and negative control tissues based on known expression patterns

• Consider cross-reactivity testing against related myosin isoforms

• Validate findings using multiple antibodies targeting different epitopes of MYH15 when possible

What are the typical applications for MYH15 antibodies in research?

MYH15 antibodies have several important applications in scientific research:

• Immunohistochemistry: Used to detect and localize MYH15 expression in tissue sections, as demonstrated in studies of human muscle spindles . This technique allows researchers to observe the regional distribution of MYH15 along intrafusal fibers and compare expression patterns between different fiber types.

• Protein expression analysis: Western blotting and similar techniques can quantify MYH15 protein levels in tissue samples.

• Genotype-phenotype correlation studies: MYH15 antibodies can help researchers investigate how genetic polymorphisms affect protein expression and localization. For instance, in studies of asthma susceptibility, MYH15 antibodies could be used to examine how polymorphisms (like rs9288876, rs7635009, and rs1454197) affect MYH15 expression in airway tissues .

• Disease mechanism investigation: Given the association between MYH15 and conditions like asthma, antibodies can be valuable tools for examining pathological changes in protein expression and distribution.

How can MYH15 antibodies be optimized for detecting specific isoforms in human muscle spindles?

Optimizing MYH15 antibodies for detecting specific isoforms in human muscle spindles requires careful consideration of several methodological aspects:

• Antibody selection: Choose antibodies that specifically recognize the MYH15 isoform rather than cross-reacting with other myosin heavy chains. Monoclonal antibodies may offer greater specificity than polyclonal alternatives.

• Tissue preparation: The detection of MYH15 in muscle spindles is highly dependent on proper tissue fixation and processing. Studies have successfully used formaldehyde-fixed, paraffin-embedded samples of biceps brachii and flexor digitorum profundus muscles .

• Antigen retrieval: Heat-induced or enzymatic antigen retrieval may be necessary to expose epitopes masked during fixation.

• Regional analysis: MYH15 expression varies along the length of intrafusal fibers, with more pronounced expression in the extracapsular region of bag fibers . Therefore, systematic sampling along the fiber length is essential for accurate characterization.

• Co-localization studies: Combine MYH15 antibodies with markers for other myosin isoforms (such as slow-tonic) to differentiate fiber types. The slow-tonic isoform has been shown to be a reliable marker for differentiating positive bag fibers from negative chain fibers .

• Controls: Include both positive controls (tissues known to express MYH15) and negative controls (tissues or fiber types known to lack MYH15 expression) in each experiment.

What are the challenges in interpreting MYH15 antibody staining patterns in human tissues?

Interpreting MYH15 antibody staining patterns presents several challenges that researchers should be aware of:

• Variable expression along fiber length: MYH15 expression varies significantly along the length of intrafusal fibers, with differences between extracapsular and intracapsular regions . This variation necessitates systematic sampling and analysis along the entire length of the fibers.

• Co-expression with other myosin isoforms: MYH15 is often co-expressed with other myosin heavy chain isoforms in the same fiber, creating complex expression patterns. For instance, bag fibers may express both MYH15 and slow-tonic isoforms, requiring careful analysis to distinguish expression patterns .

• Cross-reactivity concerns: Antibodies designed for one species may not perform identically in tissues from other species. The search results note uncertainty about whether antibodies specific for rat 2b isoform accurately reflect expression of this isoform in human bag fibers .

• Variation across muscle types: MYH15 expression patterns can differ between muscle types. Studies have examined expression in biceps brachii, flexor digitorum profundus, masseter, and laryngeal cricothyreoid muscles, finding differences in expression patterns .

• Technical variability: Different antibodies and immunohistochemical protocols may yield varying results, making standardization important for comparing findings across studies. As noted in the literature, "the estimation of expression may also depend on the antibodies utilised" .

How can MYH15 antibodies be used to investigate the relationship between MYH15 polymorphisms and asthma susceptibility?

MYH15 antibodies can be valuable tools for investigating the functional consequences of MYH15 polymorphisms associated with asthma susceptibility. A methodological approach might include:

• Tissue-specific expression analysis: Use MYH15 antibodies to examine protein expression in airway epithelium samples from individuals with different genotypes at asthma-associated loci (rs9288876, rs7635009, and rs1454197) .

• Quantitative immunohistochemistry: Measure MYH15 protein levels in tissues from patients with different genotypes to determine if polymorphisms affect protein abundance. This can be correlated with the functional findings from luciferase assays that have shown allele-dependent differences in promoter activity .

• Co-localization studies: Combine MYH15 antibodies with markers for airway inflammation or remodeling to investigate potential mechanistic links between MYH15 expression and asthma pathophysiology.

• In vitro models: Use cell culture systems to express different MYH15 variants and examine protein expression, localization, and function using MYH15 antibodies.

• Correlation with clinical parameters: Link MYH15 expression levels (detected by antibodies) with clinical measures of airway responsiveness or asthma severity in patients with different genotypes.

Research has shown that polymorphisms rs9288876, rs7635009, and rs1454197 alter transcriptional regulation of MYH15 and may be functional variants conferring susceptibility to asthma . MYH15 antibodies can help elucidate how these genetic variations translate to changes in protein expression and function in relevant tissues.

What are the optimal fixation and tissue processing methods for MYH15 immunohistochemistry?

Optimal fixation and tissue processing methods for MYH15 immunohistochemistry should be carefully selected based on the specific research objectives and tissue types:

• Fixation:

  • Formaldehyde fixation has been successfully used in studies examining MYH15 in human muscle spindles .

  • The duration of fixation should be optimized to preserve tissue morphology while maintaining antigen accessibility.

  • For some applications, fresh-frozen tissue sections may better preserve antigenicity, though with potential compromises in morphological preservation.

• Tissue processing:

  • Paraffin embedding provides excellent morphological preservation and has been used successfully in MYH15 studies .

  • Section thickness typically ranges from 5-10 μm, with thinner sections (5-7 μm) preferred for high-resolution imaging.

  • When studying muscle spindles, longitudinal sections may be more informative than cross-sections for observing regional variations in MYH15 expression along fiber length.

• Antigen retrieval:

  • Heat-induced epitope retrieval (HIER) in citrate buffer (pH 6.0) or EDTA buffer (pH 9.0) is commonly used to enhance antibody binding.

  • Optimization of retrieval conditions (temperature, duration, buffer composition) may be necessary for specific antibodies and tissue types.

• Blocking and antibody incubation:

  • Thorough blocking of non-specific binding sites using serum or protein solutions is essential.

  • Primary antibody concentrations should be titrated for each application.

  • Incubation times may vary from overnight at 4°C to shorter periods at room temperature depending on antibody characteristics.

• Detection systems:

  • Polymer-based detection systems often provide better signal-to-noise ratios than traditional ABC methods.

  • Chromogenic detection with DAB allows for permanent slides and assessment of morphology, while fluorescent detection enables multi-labeling and higher resolution imaging.

How can researchers validate the specificity of MYH15 antibodies for their experiments?

Validating the specificity of MYH15 antibodies is crucial for ensuring reliable and reproducible research results. A comprehensive validation approach should include:

• Western blot analysis:

  • Confirm that the antibody detects a protein of the expected molecular weight in tissues known to express MYH15.

  • Compare detection in positive control tissues (e.g., extraocular muscles) versus negative control tissues.

  • Assess cross-reactivity with other myosin heavy chain isoforms, particularly those with high sequence homology.

• Immunohistochemical controls:

  • Include positive control tissues with known MYH15 expression patterns.

  • Use negative control tissues or regions known not to express MYH15.

  • Perform antibody omission controls to assess non-specific binding of detection reagents.

  • Compare staining patterns with published literature on MYH15 expression.

• Competing peptide assays:

  • Pre-incubate the antibody with the immunizing peptide to block specific binding sites.

  • Compare staining with and without peptide competition to identify specific versus non-specific signals.

• Genetic validation:

  • Where possible, compare antibody staining in tissues with confirmed MYH15 expression (via mRNA analysis) versus tissues with low or no expression.

  • Consider using tissues from knockout models (if available) as negative controls.

• Multi-antibody approach:

  • Use multiple antibodies targeting different epitopes of MYH15 to confirm staining patterns.

  • Consistent results across different antibodies increase confidence in specificity.

In studies of human muscle spindles, researchers validated antibody specificity by comparing the expression patterns of multiple myosin isoforms, confirming that the slow-tonic isoform was consistently expressed in bag fibers but not chain fibers, providing an internal validation measure .

What methods can be combined with MYH15 antibody staining for comprehensive analysis of myosin isoform expression?

For comprehensive analysis of myosin isoform expression, researchers can combine MYH15 antibody staining with several complementary methods:

• Multi-label immunofluorescence:

  • Simultaneously detect multiple myosin isoforms in the same tissue section using antibodies with different species origins or isotypes.

  • Include markers for fiber types, such as slow-tonic myosin for bag fibers .

  • Incorporate structural markers to identify specific regions of muscle spindles (e.g., capsule markers).

• Serial section analysis:

  • Stain consecutive sections with different antibodies to create a comprehensive map of isoform expression.

  • This approach is particularly useful when antibody species conflicts prevent simultaneous detection.

  • Align and register images from serial sections for comparative analysis.

• mRNA analysis:

  • Complement protein detection with in situ hybridization for MYH15 mRNA.

  • Use laser capture microdissection followed by RT-PCR or RNA-seq to quantify MYH15 and other myosin transcripts in specific fiber types.

• Mass spectrometry-based proteomics:

  • Use targeted proteomics to identify and quantify specific myosin isoforms in muscle samples.

  • This approach can provide unbiased confirmation of antibody-based findings.

• Functional assays:

  • Combine expression analysis with physiological measurements of muscle fiber contractile properties.

  • Correlate MYH15 expression with specific functional characteristics of different fiber types.

• Genetic analysis:

  • Incorporate genotyping for known MYH15 polymorphisms to correlate with protein expression patterns.

  • This is particularly relevant when studying MYH15's role in conditions like asthma, where specific polymorphisms (rs9288876, rs7635009, and rs1454197) have been associated with disease risk .

Studies have shown that comprehensive analysis using multiple antibodies against different myosin isoforms can reveal complex patterns of expression. For example, research on human muscle spindles has demonstrated that bag1 and bag2 fibers can be distinguished based on isoform 1 expression patterns, with bag2 fibers consistently expressing this isoform over their entire length .

How do genetic variations in MYH15 impact antibody-detected protein expression patterns?

Genetic variations in MYH15 can significantly impact antibody-detected protein expression patterns through several mechanisms:

• Altered transcriptional regulation: Polymorphisms in promoter or enhancer regions can affect gene expression levels. For example, functional analysis of asthma-associated polymorphisms (rs9288876, rs7635009, and rs1454197) showed that these variants altered transcriptional regulation of MYH15 in luciferase reporter assays . Specifically:

  • rs9288876 allele A was associated with higher luciferase activity than allele T (P<0.001)

  • rs7635009 allele A showed lower luciferase activity than allele G (P=0.001)

  • rs1454197 allele T had lower luciferase activity than allele G (P<0.001)

• Expression level differences: When using antibodies to detect MYH15 in tissues from individuals with different genotypes, researchers might observe quantitative differences in staining intensity that reflect underlying genetic variation.

• Tissue-specific effects: Genetic variations may affect MYH15 expression differently across tissues. For instance, polymorphisms associated with airway responsiveness might show more pronounced effects in airway epithelium compared to muscle tissues .

• Epitope alterations: Some genetic variants might directly affect the protein sequence, potentially altering antibody binding sites. This could result in false negative results if the antibody's epitope is affected by the variant.

• Protein stability or localization changes: Certain variants might affect protein folding, stability, or subcellular localization, leading to differences in antibody staining patterns beyond simple changes in expression levels.

When studying the relationship between MYH15 polymorphisms and protein expression, researchers should consider genotyping samples and correlating genotypes with antibody-detected expression patterns. This approach can provide valuable insights into how genetic variation contributes to differences in MYH15 expression and function in health and disease states.

What is the evidence linking MYH15 to asthma susceptibility and how can antibodies help investigate this relationship?

The evidence linking MYH15 to asthma susceptibility comes primarily from genetic association studies, with supportive functional analyses. MYH15 antibodies can play a crucial role in further elucidating this relationship.

Evidence linking MYH15 to asthma susceptibility:

• Genetic association studies: Research in Chinese Han populations identified specific MYH15 polymorphisms associated with asthma risk :

  • The A allele of rs9288876 decreased risk of asthma (allelic model: OR=0.808, 95% CI: 0.658-0.993)

  • The G alleles of both rs7635009 and rs1454197 were associated with decreased risk of asthma under the additive model (OR=0.779, 95% CI: 0.618-0.981 and OR=0.756, 95% CI: 0.600-0.953, respectively)

• Functional analyses: Dual-luciferase reporter gene analysis demonstrated that these polymorphisms altered transcriptional regulation of MYH15, suggesting a functional basis for the association .

• Biological plausibility: MYH15 is expressed in airway epithelium, vascular endothelium, and inflammatory cells, all of which are involved in asthma pathophysiology . Previous studies also linked MYH15 variants to airway responsiveness in COPD patients, a related respiratory condition .

How antibodies can help investigate this relationship:

• Expression studies in asthma-relevant tissues:

  • Use MYH15 antibodies to compare protein expression in airway epithelium, smooth muscle, and inflammatory cells from asthmatic versus non-asthmatic individuals.

  • Examine whether expression patterns correlate with specific genotypes at asthma-associated loci.

• Cell-specific expression analysis:

  • Utilize multi-label immunohistochemistry to determine which specific cell types express MYH15 in the airways.

  • Investigate whether expression changes in response to asthma-relevant stimuli (allergens, inflammatory cytokines).

• Correlation with asthma phenotypes:

  • Analyze MYH15 expression in relation to clinical measures of airway hyperresponsiveness, inflammation, or remodeling.

  • Stratify analyses based on asthma severity or specific phenotypes.

• Mechanistic studies:

  • Use antibodies in functional assays to explore how MYH15 might contribute to cellular processes relevant to asthma (e.g., epithelial barrier function, smooth muscle contractility).

  • Develop cell models with different MYH15 variants to examine functional consequences.

• Therapeutic target validation:

  • If MYH15 emerges as a potential therapeutic target, antibodies could help validate target engagement and efficacy of interventions in preclinical models.

The evidence suggests that MYH15 may play a role in asthma susceptibility through effects on airway function, though further research is needed to fully understand the mechanisms involved .

What are the key methodological considerations when using MYH15 antibodies for studying its role in airway responsiveness?

When using MYH15 antibodies to study its role in airway responsiveness, researchers should consider several key methodological aspects:

• Tissue selection and processing:

  • Include samples representing various airway levels (trachea, bronchi, bronchioles) to capture potential regional differences in expression.

  • Consider comparing healthy versus asthmatic tissues, or tissues from individuals with different airway responsiveness.

  • Optimize fixation protocols to preserve both tissue architecture and antigenicity.

• Cell type identification:

  • Combine MYH15 antibody staining with markers for specific airway cell types (epithelial cells, smooth muscle, inflammatory cells) to determine the cellular distribution of MYH15.

  • Previous research has shown MYH15 expression in airway epithelium, vascular endothelium, and inflammatory cells , but more detailed characterization is needed.

• Genotype-expression correlation:

  • Genotype samples for known asthma-associated MYH15 polymorphisms (rs9288876, rs7635009, rs1454197) .

  • Stratify expression analyses based on genotype to investigate how genetic variation affects protein levels in relevant tissues.

• Functional assays:

  • Combine expression studies with functional measurements of airway responsiveness in appropriate models.

  • Consider in vitro approaches using primary airway cells or precision-cut lung slices that maintain tissue architecture.

• Environmental challenges:

  • Examine how MYH15 expression changes in response to asthma-relevant challenges (allergens, irritants, inflammatory mediators).

  • Determine if genetic variants affect the response to these challenges.

• Antibody validation in respiratory tissues:

  • Validate antibody specificity in airway tissues specifically, as antibody performance can vary between tissue types.

  • Include appropriate positive and negative controls.

• Quantitative assessment:

  • Develop standardized protocols for quantifying MYH15 expression in airway tissues.

  • Consider digital image analysis methods for objective quantification.

• Multi-omics integration:

  • Correlate antibody-detected protein expression with transcriptomic or epigenetic data to gain a more comprehensive understanding of MYH15 regulation in the context of airway responsiveness.

What are the emerging applications for MYH15 antibodies in respiratory and musculoskeletal research?

MYH15 antibodies are poised to play increasingly important roles in both respiratory and musculoskeletal research as our understanding of MYH15 function continues to expand. Emerging applications include:

• Precision medicine approaches for asthma:

  • Stratification of asthma patients based on MYH15 genotype and corresponding protein expression patterns.

  • Development of companion diagnostics to identify patients who might benefit from targeted therapies.

  • Investigation of MYH15 as a potential biomarker for specific asthma endotypes.

• Comprehensive mapping of muscle spindle composition:

  • Detailed characterization of myosin isoform expression patterns throughout muscle spindles using MYH15 antibodies alongside other myosin markers.

  • Investigation of how these patterns change during development, aging, or in response to exercise or injury.

  • Exploration of potential roles for MYH15 in proprioception and motor control.

• Comparative studies across species:

  • Using MYH15 antibodies to examine conservation and divergence of expression patterns across different species.

  • This could provide evolutionary insights into specialized muscle function.

• Disease mechanism investigation:

  • Beyond asthma, MYH15 expression could be relevant in other respiratory conditions involving airway remodeling or hyperresponsiveness.

  • Potential roles in neuromuscular disorders affecting proprioception or fine motor control.

• Therapeutic target validation:

  • As understanding of MYH15's role in disease progresses, antibodies will be essential tools for validating it as a potential therapeutic target.

  • Development of function-blocking antibodies could potentially serve as therapeutic prototypes.

Recent research has just begun to uncover the significance of MYH15 in human physiology and disease. The first demonstration of MYH15 and 2x isoforms in human intrafusal fibers and the first report of MYH15 gene polymorphisms' association with asthma represent significant advances that will drive future research directions.

What methodological advances would improve the utility of MYH15 antibodies in basic and clinical research?

Several methodological advances could significantly enhance the utility of MYH15 antibodies in both basic and clinical research:

• Development of isoform-specific antibodies:

  • Creation of highly specific antibodies that can distinguish MYH15 from other closely related myosin heavy chain isoforms.

  • Development of antibodies that recognize specific conformational states of MYH15 (e.g., active versus inactive forms).

• Standardized validation protocols:

  • Establishment of comprehensive validation criteria for MYH15 antibodies to ensure reproducibility across studies.

  • Creation of reference materials and positive control tissues with well-characterized MYH15 expression.

• Multiplexed detection systems:

  • Development of multiplex immunoassays allowing simultaneous detection of MYH15 alongside other relevant proteins.

  • Integration with spatial transcriptomics approaches to correlate protein expression with mRNA levels at single-cell resolution.

• Quantitative imaging approaches:

  • Standardized protocols for quantitative immunohistochemistry of MYH15.

  • Machine learning algorithms for automated analysis of staining patterns and intensity across tissues.

• In vivo imaging capabilities:

  • Development of labeled antibody fragments or mimetics suitable for in vivo imaging.

  • This could allow non-invasive monitoring of MYH15 expression in animal models of disease.

• Functional antibodies:

  • Creation of antibodies that not only detect MYH15 but also modulate its function.

  • These could serve as valuable research tools and potential therapeutic prototypes.

• Antibodies recognizing genetic variants:

  • Development of antibodies specifically recognizing products of disease-associated MYH15 variants.

  • This would be particularly valuable for studying how genetic polymorphisms affect protein structure and function.

• Integration with proteomics approaches:

  • Use of MYH15 antibodies in immunoprecipitation followed by mass spectrometry to identify interaction partners.

  • This could provide insights into the functional role of MYH15 in health and disease.

Given that the knowledge about MYH15's role in human physiology and disease is still emerging, development of these methodological advances would significantly accelerate research progress in this field.

How might future research on MYH15 bridge the gap between genetic associations and functional mechanisms in respiratory diseases?

Bridging the gap between genetic associations and functional mechanisms of MYH15 in respiratory diseases will require integrated approaches that connect genetic variation to molecular, cellular, and physiological outcomes. Future research strategies might include:

• Comprehensive genotype-phenotype correlation studies:

  • Large-scale studies examining the relationship between MYH15 polymorphisms, protein expression (using antibodies), and clinical parameters of respiratory function.

  • Expansion beyond the currently identified polymorphisms (rs9288876, rs7635009, rs1454197) to examine the full spectrum of genetic variation in MYH15.

• Mechanistic studies in relevant cell types:

  • Development of primary airway epithelial cell cultures from donors with different MYH15 genotypes.

  • CRISPR-based editing to introduce or correct specific MYH15 variants in relevant cell models.

  • Assessment of cellular functions relevant to asthma pathophysiology (barrier function, mucus production, inflammatory responses).

• Animal models with humanized MYH15 variants:

  • Generation of transgenic models expressing human MYH15 variants to study their impact on airway function in vivo.

  • Examination of how these variants affect responses to asthma-relevant challenges.

• Multi-omics integration:

  • Combination of genomics, transcriptomics, proteomics, and metabolomics data to create comprehensive models of how MYH15 variants influence respiratory function.

  • Analysis of epigenetic modifications that might mediate between genetic variation and expression changes.

• Structural biology approaches:

  • Determination of how MYH15 polymorphisms affect protein structure and function.

  • This could provide molecular-level insights into functional consequences of genetic variation.

• Clinical translation:

  • Development of genotype-based stratification approaches for clinical trials in asthma.

  • Investigation of whether MYH15 status predicts response to specific asthma therapies.

• Systems biology modeling:

  • Integration of data into computational models that predict how MYH15 variants influence airway physiology.

  • These models could generate testable hypotheses about intervention points.

• Longitudinal studies:

  • Examination of how MYH15 expression changes over time in relation to disease progression.

  • Investigation of potential interactions between genetic predisposition and environmental exposures.

The research linking MYH15 polymorphisms to asthma is relatively recent , suggesting that we are just beginning to understand its role in respiratory diseases. Future studies using MYH15 antibodies as key research tools will be essential for elucidating the functional mechanisms underlying these genetic associations.

What are the recommended protocols for Western blot analysis using MYH15 antibodies?

When performing Western blot analysis using MYH15 antibodies, researchers should consider the following optimized protocol:

• Sample preparation:

  • Extract proteins from tissues known to express MYH15 (e.g., muscle tissue, airway epithelium) using appropriate lysis buffers.

  • Include protease inhibitors to prevent degradation of MyHC proteins, which are susceptible to proteolysis.

  • For muscle tissues, special extraction buffers designed for myofibrillar proteins may improve yields.

• Protein quantification and loading:

  • Accurately quantify protein concentration using methods like BCA or Bradford assay.

  • Load 20-50 μg of total protein per lane, depending on MYH15 abundance in the sample.

  • Include positive control samples with known MYH15 expression.

• Gel electrophoresis:

  • Use low percentage (6-8%) SDS-PAGE gels to effectively resolve high molecular weight myosin heavy chains (~200-220 kDa).

  • Consider gradient gels (4-15%) when analyzing MYH15 alongside other proteins of interest.

  • Run the gel at lower voltage (80-100V) to improve resolution of high molecular weight proteins.

• Transfer conditions:

  • Use wet transfer systems for efficient transfer of high molecular weight proteins.

  • Extend transfer time (overnight at low voltage or 2-3 hours at higher voltage) with cooling.

  • Consider adding SDS (0.1%) to the transfer buffer to improve movement of large proteins.

• Blocking and antibody incubation:

  • Block membranes with 5% non-fat dry milk or BSA in TBST.

  • Incubate with optimized dilution of MYH15 primary antibody (typically 1:500 to 1:2000, but should be determined empirically).

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

• Detection and analysis:

  • Use high-sensitivity detection methods due to potentially low expression levels of MYH15 in some tissues.

  • Include molecular weight markers to confirm band size (~200-220 kDa for MyHC proteins).

  • Perform densitometric analysis to quantify relative expression levels.

• Controls and validation:

  • Include loading controls appropriate for the tissue type (e.g., GAPDH, β-actin, total protein stain).

  • Run samples from tissues known to lack MYH15 expression as negative controls.

  • Consider antibody validation using competing peptide approaches.

When analyzing Western blot results, researchers should be aware that myosin heavy chain isoforms have similar molecular weights, so band position alone may not definitively identify MYH15. Complementary approaches, such as immunoprecipitation followed by mass spectrometry, may be needed for absolute confirmation in complex samples.

What are the key considerations for immunohistochemical detection of MYH15 in different tissue types?

Immunohistochemical detection of MYH15 requires careful optimization based on tissue type and research objectives. Key considerations include:

• Tissue-specific optimization:

  For muscle tissues:

  • Longitudinal sections are preferred for muscle spindles to observe regional variations along intrafusal fibers .

  • Consider fiber orientation when interpreting staining patterns, as MYH15 expression varies between extracapsular and intracapsular regions of bag fibers .

  • Use serial sections to compare MYH15 distribution with other myosin isoforms, particularly slow-tonic myosin which serves as a marker for bag fibers .

  For airway tissues:

  • Ensure sections capture the full airway wall, including epithelium, smooth muscle, and inflammatory cells.

  • Consider the airway level (trachea, bronchi, bronchioles) when interpreting results, as expression may vary.

  • Include bronchial-associated lymphoid tissue when examining MYH15 in inflammatory cells.

• Antibody selection and validation:

  • Choose antibodies with demonstrated specificity for human MYH15.

  • Validate antibodies in each tissue type, as performance can vary across tissues.

  • Consider using multiple antibodies targeting different epitopes to confirm findings.

• Antigen retrieval optimization:

  • Optimize antigen retrieval methods for each tissue type, as requirements may differ.

  • For muscle tissues, heat-induced epitope retrieval in citrate buffer (pH 6.0) is often effective.

  • For airway tissues, EDTA-based retrieval (pH 9.0) may provide better results for some antibodies.

• Detection system selection:

  • Choose detection systems based on expected expression levels and required sensitivity.

  • Tyramide signal amplification may be helpful for detecting low-abundance expression.

  • For co-localization studies, select fluorescent systems with minimal spectral overlap.

• Interpretation guidelines:

  • Establish clear criteria for positive staining based on expected subcellular localization.

  • For muscle tissues, note that MYH15 expression varies along fiber length and between fiber types .

  • For airway tissues, distinguish between expression in epithelial, mesenchymal, and inflammatory cells.

• Quantification approaches:

  • Develop standardized methods for quantifying MYH15 expression.

  • Consider both staining intensity and the percentage of positive cells.

  • Digital image analysis can provide objective quantification of expression patterns.

• Controls:

  • Include appropriate positive and negative tissue controls in each experiment.

  • For muscle studies, slow-tonic myosin positive/negative fibers can serve as internal controls .

  • Include isotype controls to assess non-specific binding.

Research has shown that MYH15 expression varies significantly between different regions of intrafusal fibers and across different muscle spindles and muscles . This heterogeneity should be considered when designing and interpreting immunohistochemical studies.

How can researchers troubleshoot common issues with MYH15 antibody applications?

Researchers may encounter various challenges when working with MYH15 antibodies. The following troubleshooting guide addresses common issues and their solutions:

Western Blot Issues:

• No bands or weak signal:

  • Cause: Insufficient protein loading, inadequate antibody concentration, or low MYH15 expression.

  • Solution: Increase protein loading (50-100 μg), optimize antibody concentration, extend exposure time, use more sensitive detection methods, or confirm MYH15 expression in the tissue via other methods.

• Multiple bands:

  • Cause: Cross-reactivity with other myosin isoforms, protein degradation, or non-specific binding.

  • Solution: Use more specific antibodies, include protease inhibitors during sample preparation, optimize blocking conditions, and validate with competing peptide approaches.

• Unexpected band size:

  • Cause: Post-translational modifications, splice variants, or non-specific binding.

  • Solution: Compare with positive control tissues, verify with mass spectrometry, or use antibodies targeting different epitopes.

Immunohistochemistry Issues:

• Weak or absent staining:

  • Cause: Inadequate antigen retrieval, low antibody concentration, or fixation-related epitope masking.

  • Solution: Optimize antigen retrieval conditions (method, buffer, time, temperature), increase antibody concentration, extend incubation time, or use amplification systems.

• High background staining:

  • Cause: Insufficient blocking, excessive antibody concentration, or endogenous peroxidase/biotin activity.

  • Solution: Optimize blocking (longer time, different blocking agents), dilute primary antibody, include additional washing steps, or block endogenous activities.

• Inconsistent staining patterns:

  • Cause: Tissue fixation variability, regional expression differences, or technical inconsistency.

  • Solution: Standardize fixation protocols, systematically sample along muscle fibers to account for regional variation , and implement rigorous quality control measures.

General Validation Issues:

• Discrepancies between antibodies:

  • Cause: Different epitopes, varying specificities, or technical factors.

  • Solution: Compare with functional data, use complementary techniques (RNA expression, mass spectrometry), and consider the limitations of each antibody.

• Conflicting results with literature:

  • Cause: Different antibodies, tissues, or methodologies.

  • Solution: Note that "the revealed pattern of isoform co-expression only partially agrees with the results of previous, more extensive studies" , suggesting variation is expected. Carefully document methodological differences and consider validation with multiple approaches.

• Difficulties distinguishing MYH15 from other isoforms:

  • Cause: High sequence homology between myosin isoforms.

  • Solution: Use co-staining with antibodies against other isoforms (e.g., slow-tonic as a marker for bag fibers ), perform careful antibody validation, and consider complementary molecular techniques.

• Genotype-dependent staining variations:

  • Cause: Genetic polymorphisms affecting protein expression or antibody binding.

  • Solution: Genotype samples for known polymorphisms (rs9288876, rs7635009, rs1454197) , stratify analysis by genotype, and consider using antibodies targeting conserved regions.

When troubleshooting, remember that MYH15 expression shows natural variation "along their length, across different muscle spindles and muscles" , which may complicate interpretation of results. A systematic approach to validation and consistency in technical methods is essential for reliable results.

What are the most informative publications for researchers using MYH15 antibodies?

For researchers working with MYH15 antibodies, several key publications provide valuable methodological insights and contextual information:

• MYH15 expression in muscle spindles:

  • The study by Mazzotti et al. (2023) provides detailed immunohistochemical characterization of MyHC-15 expression in human muscle spindles, describing its distribution in different regions of intrafusal fibers . This paper offers important methodological details on antibody selection and validation for muscle tissue applications.

• MYH15 polymorphisms and asthma:

  • The research by Wang et al. (2019) represents the first investigation of MYH15 polymorphisms in relation to asthma susceptibility . This study provides context for researchers interested in respiratory applications of MYH15 antibodies and includes functional analysis of how specific polymorphisms affect gene expression.

• MYH15 and airway responsiveness:

  • The work by Hansel et al. (2015) (referenced in the search results but not directly provided) linked MYH15 polymorphisms to airway responsiveness in COPD patients and demonstrated MYH15 protein expression in airway epithelium . This study provides important background for researchers investigating respiratory applications.

• General myosin heavy chain immunohistochemistry:

  • While not specific to MYH15, comprehensive reviews of myosin heavy chain immunohistochemistry in human muscles provide valuable methodological guidance applicable to MYH15 studies.

• Functional studies of myosin heavy chains:

  • Publications examining the functional properties of various myosin heavy chain isoforms provide important context for interpreting MYH15 expression patterns.

When accessing these resources, researchers should note that the field is still developing, with the first demonstration of MYH15 in human intrafusal fibers being relatively recent . Therefore, staying updated with new methodological developments is essential.

What online resources and databases are available for MYH15 antibody validation and experimental design?

Researchers working with MYH15 antibodies can leverage several online resources and databases to support antibody validation and experimental design:

• Antibody validation databases:

  • Antibodypedia: Provides user-submitted validation data for commercial antibodies, including applications and recommended protocols.

  • The Human Protein Atlas: Contains immunohistochemistry data for many proteins, though MYH15 coverage may be limited.

  • CiteAb: Aggregates antibody citations from scientific literature, helping researchers identify antibodies with proven performance in published studies.

• Protein and gene databases:

  • UniProt: Provides comprehensive information about MYH15 protein structure, domains, and post-translational modifications.

  • Ensembl/NCBI Gene: Offers genomic context, transcript variants, and cross-species conservation data for MYH15.

  • GTEx Portal: Contains tissue-specific expression data for MYH15 at the mRNA level, which can guide tissue selection for protein studies.

• Genetic variation resources:

  • dbSNP/1000 Genomes Project: Catalogs genetic variations in MYH15, including the polymorphisms associated with asthma susceptibility (rs9288876, rs7635009, and rs1454197) .

  • GnomAD: Provides population frequency data for MYH15 variants, which can inform the interpretation of genetic findings.

• Protocol repositories:

  • Protocols.io: Contains detailed protocols for various antibody applications that can be adapted for MYH15 studies.

  • Bio-protocol: Peer-reviewed protocols that may include relevant immunohistochemistry or Western blot methodologies.

• Method optimization tools:

  • BLAST: For analyzing sequence homology between MYH15 and other myosin heavy chains to predict potential cross-reactivity.

  • Epitope prediction tools: Help identify likely antibody binding sites and potential issues with genetic variants.

• Structural databases:

  • Protein Data Bank (PDB): May contain structural information for myosin heavy chains that could inform understanding of MYH15.

  • AlphaFold DB: Provides AI-predicted protein structures that might include MYH15 or closely related proteins.

• Disease association databases:

  • GWAS Catalog: Contains information on genome-wide association studies that may include MYH15 variants.

  • PhenoScanner: Allows researchers to examine phenotype associations for specific MYH15 variants.

When using these resources, researchers should be aware that MYH15 is a relatively understudied protein compared to other myosin heavy chains, so information may be limited in some databases. Cross-validation using multiple resources is recommended for robust experimental design.

What are the recommended quality control standards for purchasing and using commercial MYH15 antibodies?

When purchasing and using commercial MYH15 antibodies, researchers should apply rigorous quality control standards to ensure reliable and reproducible results:

Selection Criteria for Commercial Antibodies:

• Validation documentation:

  • Request comprehensive validation data from manufacturers, including Western blot images, immunohistochemistry results, and specificity testing.

  • Prioritize antibodies validated in human tissues relevant to your research (muscle spindles for musculoskeletal research , airway tissues for respiratory studies ).

  • Look for evidence of testing in multiple applications (Western blot, IHC, IF, etc.) if you plan to use the antibody in different contexts.

• Epitope information:

  • Select antibodies with clearly defined epitopes.

  • Consider the epitope location relative to known MYH15 polymorphisms, particularly if studying genetic variants associated with asthma (rs9288876, rs7635009, rs1454197) .

  • For specific applications, choose antibodies targeting functional domains relevant to your research question.

• Clone type and species reactivity:

  • For highly specific detection, consider monoclonal antibodies targeting unique MYH15 epitopes.

  • For broader detection of potentially modified forms, polyclonal antibodies may be advantageous.

  • Verify species reactivity is appropriate for your samples (human, mouse, rat, etc.).

• Publication record:

  • Prioritize antibodies cited in peer-reviewed publications, especially those studying MYH15 in contexts similar to your research.

  • Review published methods sections for application-specific details.

In-house Validation Requirements:

• Positive and negative controls:

  • Test antibodies on tissues known to express MYH15 (e.g., extraocular muscles, certain intrafusal fibers in muscle spindles ).

  • Include negative control tissues where MYH15 is not expected to be expressed.

  • For muscle studies, exploit the differential expression pattern (e.g., expression in bag fibers but not chain fibers) as an internal validation.

• Specificity testing:

  • Perform peptide competition assays where the immunizing peptide blocks specific binding.

  • Compare results with alternative antibodies targeting different MYH15 epitopes.

  • Correlation with mRNA expression data provides additional validation.

• Reproducibility assessment:

  • Test lot-to-lot consistency if using the antibody over extended research periods.

  • Establish standardized protocols with defined acceptance criteria.

  • Include standard samples across experiments to monitor performance over time.

• Application-specific validation:

  • For Western blot: Verify the antibody detects a protein of the expected molecular weight.

  • For IHC/IF: Confirm appropriate subcellular localization and expression patterns consistent with literature.

  • For flow cytometry: Validate using cells with known MYH15 expression profiles.

• Documentation practices:

  • Maintain detailed records of antibody information (catalog number, lot number, host species, clonality, epitope).

  • Document all validation experiments, including images of positive and negative controls.

  • Record optimization parameters for each application (dilutions, incubation times, detection methods).