MYOM3 Antibody

What is MYOM3 and what is its role in muscle physiology?

MYOM3 (myomesin-3) is a structural component of the M-band in striated muscle with a calculated molecular weight of 162 kDa. It belongs to a family of closely related structural proteins, including MYOM1, MYOM2 (or M protein), and MYOM3, which are detected at the M-band of the sarcomere in striated skeletal muscles. MYOM3 functions primarily in sarcomere stability and resistance during intense or sustained stretching, making it crucial for maintaining muscle integrity during contraction and relaxation cycles . The protein is predominantly expressed in intermediate-speed fibers of skeletal muscle, suggesting a tissue-specific function that may be important when considering antibody applications in different muscle types .

How is MYOM3 structurally organized and what domains are important for antibody targeting?

MYOM3 is a protein of 1437 amino acids (162.2 kDa), organized in a domain structure similar to other myomesin family members. The protein contains multiple domains that can be targeted by antibodies, with commercially available antibodies often targeting specific regions such as amino acids 887-1178 or 430-512 . When designing experiments, researchers should consider that fragments of MYOM3 rather than the intact protein may be detected in biological samples. Western blot analysis of serum samples has revealed the presence of two characteristic MYOM3 fragments of approximately 100 kDa and 130 kDa, which have similar C-terminal ends but different N-termini .

What makes MYOM3 particularly relevant for muscular dystrophy research?

MYOM3 has emerged as a potential biomarker for muscular dystrophies, particularly Duchenne muscular dystrophy (DMD) and limb-girdle muscular dystrophy type 2D (LGMD2D). High-resolution mass spectrometry studies have identified MYOM3 fragments in sera of patients with these conditions . Importantly, MYOM3 fragments demonstrate lower inter-individual variations compared to the commonly used creatine kinase (CK) assay, making them potentially more reliable biomarkers. In animal models, these fragments have shown superior properties for early disease detection and are less sensitive to physical exercise compared to CK measurements .

What species reactivity can be expected from commercially available MYOM3 antibodies?

Commercial MYOM3 antibodies typically show cross-reactivity with human, mouse, and rat samples, making them versatile tools for comparative studies across these species . This cross-reactivity is supported by the high sequence homology between species, with recombinant protein controls showing approximately 77% sequence identity between human MYOM3 and mouse/rat orthologs in specific regions . When planning experiments involving other species, researchers should perform validation studies or consult literature for confirmed reactivity.

How should researchers properly store and handle MYOM3 antibodies to maintain reactivity?

MYOM3 antibodies are typically provided in liquid form in a buffer containing PBS with 0.02% sodium azide and 50% glycerol at pH 7.3 . For optimal stability, antibodies should be stored at -20°C, where they remain stable for one year after shipment. Aliquoting is generally unnecessary for -20°C storage. Some preparations may contain 0.1% BSA as a stabilizer . Before each use, antibodies should be thawed completely and mixed gently to ensure homogeneity.

How should researchers design validation experiments to confirm MYOM3 antibody specificity?

When validating MYOM3 antibody specificity, researchers should implement a multi-faceted approach. First, blocking experiments should be conducted using recombinant protein fragments, such as Human MYOM3 (aa 430-512) Control Fragment. For optimal blocking, pre-incubate the antibody with a 100x molar excess of the protein fragment control for 30 minutes at room temperature before application . Additionally, researchers should include positive controls (skeletal muscle tissue) where MYOM3 is highly expressed, and negative controls (tissues with minimal MYOM3 expression) in their validation protocol. Western blot analysis should demonstrate bands at the expected molecular weights (approximately 162 kDa for full-length protein, and 100 kDa and 130 kDa for the fragments commonly observed in serum samples) .

What methodological considerations are important when using MYOM3 antibodies for detecting MYOM3 fragments in serum samples?

Detection of MYOM3 fragments in serum requires careful methodological attention. When performing western blot analysis, researchers should use antibodies targeting specific regions, such as amino acids 887-1178, which have been validated for detecting the characteristic 100 kDa and 130 kDa fragments . For immunoprecipitation and subsequent mass spectrometry analysis, it's important to note that these fragments have similar C-terminal ends but different N-termini. Based on previous research, the larger fragment contains peptides covering amino acids 254-1331, while the smaller fragment contains peptides covering amino acids 476-1331 . Sample preparation is also critical - researchers may need to consider immunodepletion of abundant serum proteins to improve detection sensitivity, similar to approaches used in comprehensive proteomic studies .

What are the potential pitfalls and troubleshooting strategies when working with MYOM3 antibodies in complex samples?

When working with complex samples like serum or tissue homogenates, researchers may encounter several challenges. First, the presence of highly abundant proteins in serum can mask MYOM3 detection. This can be addressed through immunodepletion of major serum proteins, as demonstrated in studies where the 12 most abundant proteins were removed . Second, the detection of specific MYOM3 fragments rather than the full-length protein may complicate analysis if antibodies don't recognize the relevant epitopes. Researchers should select antibodies with epitopes present in the fragments of interest . Finally, cross-reactivity with other myomesin family members (MYOM1, MYOM2) due to sequence homology may occur. This can be addressed by including appropriate controls and confirmation with mass spectrometry when possible .

How do MYOM3 fragments compare to traditional biomarkers like creatine kinase in muscular dystrophy research?

MYOM3 fragments offer several advantages over traditional biomarkers like creatine kinase (CK) in muscular dystrophy research. Comprehensive studies have shown that MYOM3 fragments demonstrate lower inter-individual variability compared to CK, making them potentially more reliable biomarkers . In animal models of dystrophin deficiency (mdx mice), MYOM3 fragments were more reliable for early disease detection and less sensitive to physical exercise when compared to CK . Furthermore, MYOM3 fragments showed superior correlation with the restoration of the dystrophin-associated protein complex (DAPC) and physical force recovery following gene therapy in LGMD2D mouse models . This suggests that MYOM3 fragments may provide more accurate assessment of disease progression and treatment efficacy.

What is the protocol for quantitative assessment of MYOM3 fragments in patient samples?

For quantitative assessment of MYOM3 fragments in patient samples, researchers should implement a standardized protocol. Based on published research, western blot analysis using antibodies targeting amino acids 887-1178 of MYOM3 can effectively detect the characteristic 100 kDa and 130 kDa fragments . Sample preparation should include careful handling to minimize protein degradation, potentially including protease inhibitors. For more precise quantification, researchers may employ mass spectrometry-based approaches. In previous studies, a comprehensive high-resolution mass spectrometry method was used, with samples organized into age-matched groups and pooled to reduce variability . When analyzing results, researchers should focus on the relative abundance of the specific fragments rather than the full-length protein, as these fragments are the relevant biomarkers in serum samples.

How do different muscular dystrophies affect the expression pattern and fragmentation of MYOM3?

Research has shown that MYOM3 fragments are present at elevated levels in different types of muscular dystrophies, including Duchenne muscular dystrophy (DMD) and limb-girdle muscular dystrophy type 2D (LGMD2D) . While the specific fragmentation patterns appear similar across these conditions (producing fragments of approximately 100 kDa and 130 kDa), the absolute levels and dynamics may vary between different muscular dystrophies. In therapeutic model systems, such as dystrophin restoration by antisense oligonucleotide-mediated exon-skipping in mdx mice and α-sarcoglycan expression restoration in KO-SGCA mice, MYOM3 fragment levels decreased toward wild-type levels in a dose-dependent manner following treatment . This suggests that the fragmentation process is directly related to muscle damage and turnover of sarcomeric proteins, regardless of the specific molecular defect causing the dystrophy.

What antigen retrieval methods are optimal for MYOM3 detection in tissue sections?

For optimal MYOM3 detection in tissue sections using immunohistochemistry (IHC) or immunofluorescence (IF), specific antigen retrieval methods are recommended. Based on antibody validation data, TE buffer at pH 9.0 is suggested as the primary antigen retrieval method for MYOM3 detection in skeletal muscle tissue sections . Alternatively, citrate buffer at pH 6.0 may also be used for antigen retrieval, though this may produce different sensitivity levels . These retrieval methods help expose epitopes that might be masked during fixation processes, particularly in formalin-fixed, paraffin-embedded (FFPE) samples. Researchers should optimize the retrieval time and temperature based on their specific sample preparation methods, as over-retrieval can lead to tissue damage while under-retrieval may result in weak signal.

How should researchers design experiments to monitor MYOM3 as a biomarker in therapeutic intervention studies?

When designing experiments to monitor MYOM3 as a biomarker in therapeutic intervention studies, researchers should implement a longitudinal approach with careful consideration of sampling timepoints. Based on previous research in therapeutic model systems, MYOM3 fragment levels in serum can reflect treatment efficacy and correlate with functional improvements . Researchers should collect baseline samples before intervention, followed by regular intervals during treatment. In gene therapy studies using viral vectors, a dose-dependent relationship between the therapeutic agent and MYOM3 restoration has been observed, suggesting that multiple dosage groups should be included in experimental design . Importantly, MYOM3 fragment analysis should be combined with functional assessments (e.g., muscle force measurements) and molecular analyses (e.g., restoration of dystrophin-associated protein complex) to establish correlations between biomarker levels and physiological outcomes .

What control samples and normalization methods are recommended for accurate MYOM3 quantification?

For accurate MYOM3 quantification, appropriate control samples and normalization methods are essential. In serum biomarker studies, age-matched healthy controls are crucial, as baseline levels may vary with age . When analyzing MYOM3 in tissue samples, researchers should include both positive controls (skeletal muscle tissue) and negative controls (tissues with minimal MYOM3 expression) in each experimental run . For western blot experiments, loading controls appropriate for serum samples should be used, and multiple technical replicates are recommended to ensure reproducibility. When comparing MYOM3 levels across different disease states or treatment conditions, normalization to total protein rather than individual housekeeping proteins may provide more accurate results, particularly in serum samples where traditional housekeeping proteins may not be appropriate . For mass spectrometry-based quantification, label-free protein quantification with appropriate normalization algorithms should be employed to account for technical variations .