MYH2 Antibody

What is MYH2 and why is it significant in muscle research?

MYH2 (myosin heavy chain 2) is a critical protein component of skeletal muscle with a molecular weight of approximately 223 kDa and 1941 amino acid residues in humans. It localizes to the cytoplasm and exists in up to two different isoforms. As a member of the myosin protein family, MYH2 plays an essential role in muscle contraction, making it a significant target for understanding muscle physiology and pathology. MYH2 is predominantly expressed in type IIa fast-twitch muscle fibers, which balance power and endurance capabilities .

The protein is also known by several synonyms, including IBM3, MYH2A, MYHSA2, MYHas8, MYPOP, MyHC-2A, MyHC-IIa, and CMYP6. Importantly, mutations in the MYH2 gene have been associated with various myopathies, making it a valuable target for both basic muscle biology and disease-related research . MYH2 participates in complex interactions with actin (ACTA1) and regulatory proteins like tropomyosin and troponin, which control muscle contraction in response to calcium signaling .

What are the standard applications for MYH2 antibodies in research protocols?

MYH2 antibodies are versatile research tools that can be employed across multiple experimental techniques. The most common applications include:

• Western Blot (WB): For quantitative analysis of MYH2 protein expression in muscle lysates. This technique is particularly valuable for studying changes in MYH2 expression during myogenic differentiation or disease states .

• Immunofluorescence (IF): Used to visualize MYH2 distribution within skeletal muscle tissues or differentiated myotubes. This approach allows researchers to analyze fiber type composition and morphological features of muscle cells .

• Immunohistochemistry (IHC): For detection of MYH2 in paraffin-embedded or frozen tissue sections, enabling analysis of fiber type distribution in muscle biopsies .

• Immunoprecipitation (IP): To isolate MYH2 and its binding partners, facilitating the study of protein-protein interactions within the contractile apparatus .

• ELISA: For quantitative measurement of MYH2 concentrations in biological fluids, tissue homogenates, or cell culture supernatants .

Over 600 citations in the scientific literature document the use of MYH2 antibodies, with Western Blot being particularly prevalent among researchers studying skeletal muscle biology .

How can researchers distinguish between different myosin heavy chain isoforms?

Distinguishing between myosin heavy chain isoforms requires careful selection of antibodies and experimental conditions. Here's a methodological approach:

• Isoform-specific antibodies: Select antibodies that specifically recognize MYH2 (MyHC-IIa) without cross-reactivity to other isoforms like MYH1 (MyHC-IIx) or MYH7 (MyHC-I/β). Monoclonal antibodies such as clone A4.74 are designed to specifically detect MYH2 .

• Immunofluorescence with co-staining: Perform dual or triple immunofluorescence labeling using antibodies against different myosin isoforms (e.g., MYH7 for slow fibers and MYH1/MYH2 for fast fibers). This approach allows visual discrimination of fiber types based on their myosin content .

• Electrophoretic separation: Utilize SDS-PAGE with specific conditions (e.g., low percentage gels, extended run times) that can separate the different myosin heavy chain isoforms based on their slightly different molecular weights.

• qPCR validation: Complement protein-level studies with mRNA analysis using isoform-specific primers to confirm expression patterns of different myosin genes .

When performing immunohistochemistry, researchers should be aware that some antibodies recognize multiple fast myosin isoforms, while others are highly specific. The search results indicate that antibodies like clone MF20 recognize the heavy chain of myosin II, while others can distinguish between slow MYH7 and fast MYH1/MYH2 fibers .

How can MYH2 antibodies be utilized to study myogenic differentiation?

MYH2 antibodies provide powerful tools for investigating myogenic differentiation in various experimental models. Methodologically, researchers can:

• Time-course analysis: Monitor MYH2 expression at different time points during differentiation (e.g., 48h, 96h) to track myotube formation and maturation. This approach allows correlation of MYH2 expression with other myogenic markers like MyoD and MyoG .

• Differentiation index calculation: Quantify the percentage of nuclei incorporated into MYH2-positive myotubes to assess differentiation efficiency. This calculation provides a standardized measure that can be compared across experimental conditions .

• Fusion index determination: Calculate the number of nuclei per MYH2-positive myotube to evaluate myoblast fusion capacity, categorizing myotubes based on nuclear content (e.g., 1-3 nuclei, >4 nuclei) .

• Co-analysis with regulatory pathways: Combine MYH2 immunostaining with analysis of signaling pathways (e.g., PI3K-AKT-mTOR) to understand molecular mechanisms regulating myogenesis. This approach has revealed that phosphorylation of PI3K (p85 alpha), AKT (ser473), and mTOR (ser2448) correlates with MYH2 expression during differentiation .

• Interference studies: Use siRNA or miRNA approaches to manipulate gene expression (e.g., BAMBI, miR-106a-5p) and assess effects on myogenic differentiation through MYH2 expression analysis .

The search results demonstrate that MYH2 immunostaining with DAPI counterstaining provides clear visualization of multinucleated myotubes, enabling quantitative assessment of myogenic differentiation under various experimental conditions .

What methodological considerations are important when optimizing Western blot protocols for MYH2 detection?

Detecting MYH2 by Western blot presents unique challenges due to its high molecular weight (223 kDa) and abundance in muscle tissues. Researchers should consider these methodological approaches:

• Sample preparation optimization:

  • Use specialized lysis buffers containing high salt concentrations (>300mM NaCl) to efficiently extract myofibrillar proteins

  • Include protease inhibitors to prevent degradation of the large MYH2 protein

  • Sonicate samples briefly to shear DNA and reduce viscosity

• Gel electrophoresis parameters:

  • Utilize low percentage acrylamide gels (6-8%) to effectively resolve the high molecular weight MYH2

  • Extend running time at lower voltage (80-100V) to achieve better separation from other myosin isoforms

  • Consider gradient gels (4-15%) for simultaneously analyzing MYH2 and smaller proteins like MyoD

• Transfer conditions:

  • Implement wet transfer methods rather than semi-dry for efficient transfer of large proteins

  • Use lower current settings with extended transfer times (overnight at 30V)

  • Add SDS (0.1%) to the transfer buffer to facilitate the movement of large proteins

• Detection strategy:

  • Employ high-sensitivity detection systems to visualize bands clearly

  • Use appropriate loading controls specific for muscle tissue (e.g., GAPDH, α-tubulin)

  • Consider stronger blocking solutions (5% BSA) to reduce background caused by the abundance of MYH2

When analyzing MYH2 expression changes during myogenic differentiation, researchers should collect samples at optimal time points (typically 96h post-differentiation for C2C12 cells) when MYH2 expression is robust enough for reliable detection .

How can researchers effectively differentiate between healthy and pathological muscle using MYH2 antibodies?

MYH2 antibodies can be instrumental in distinguishing normal from pathological muscle tissue through several methodological approaches:

• Fiber type distribution analysis:

  • Perform immunohistochemistry or immunofluorescence to quantify the proportion of MYH2-positive fibers

  • Compare fiber type distribution between patient samples and healthy controls

  • Identify abnormal fiber type grouping that may indicate reinnervation patterns

• Morphometric assessment:

  • Measure cross-sectional area of MYH2-positive fibers to detect atrophy or hypertrophy

  • Evaluate fiber shape and internal nuclei positioning as indicators of myopathic changes

  • Quantify fiber size variability coefficient as a measure of pathological change

• Protein expression quantification:

  • Use Western blot or ELISA to measure absolute MYH2 protein levels in muscle homogenates

  • Compare MYH2:total protein ratios between healthy and diseased samples

  • Assess changes in the relative abundance of different myosin isoforms

• Structural analysis:

  • Combine MYH2 staining with markers of sarcomeric organization to assess structural integrity

  • Evaluate subcellular localization of MYH2 to detect abnormal accumulation or depletion

MYH2 gene mutations have been linked to specific myopathies, making MYH2 antibodies valuable tools for diagnostic research . Importantly, search results showed that when comparing muscle progenitor cells from different sources, researchers found no significant differences in fusion coefficient or myotube morphology based on MYH2 staining, indicating the importance of quantitative analysis beyond visual inspection .

How can researchers address cross-reactivity issues with MYH2 antibodies?

Cross-reactivity among myosin heavy chain isoforms presents a significant challenge for researchers. To minimize this issue:

• Antibody selection and validation:

  • Choose monoclonal antibodies with documented specificity for MYH2 (e.g., clone A4.74)

  • Verify specificity using samples with known myosin isoform expression profiles

  • Consider using recombinant MYH2 protein as a positive control to confirm specificity

• Experimental controls:

  • Include tissues with predominant expression of specific myosin isoforms (e.g., soleus for MYH7, EDL for MYH1/MYH2)

  • Use knockout or knockdown models when available to confirm antibody specificity

  • Perform parallel experiments with mRNA assessment to correlate with protein detection

• Optimization strategies:

  • Titrate antibody concentrations to minimize non-specific binding

  • Modify blocking solutions (e.g., use casein instead of BSA) to reduce background

  • Adjust incubation times and temperatures to enhance specificity

• Pre-absorption controls:

  • Pre-incubate the antibody with purified MYH2 protein to confirm that staining is eliminated

  • Compare staining patterns with multiple antibodies targeting different epitopes of MYH2

The search results indicate that some antibodies recognize multiple myosin isoforms, while others are highly specific. For example, the A4.74 monoclonal antibody is designed to specifically detect MYH2 in multiple species including mouse, rat, human, and rabbit tissues .

What are the optimal sample preparation protocols for MYH2 immunohistochemistry?

Successful MYH2 immunohistochemistry requires careful attention to sample preparation. Based on the search results, researchers should consider:

• Fixation methods:

  • For paraffin embedding: Use 10% neutral buffered formalin fixation for 24-48 hours

  • For frozen sections: Utilize fresh-frozen tissue preserved in isopentane cooled in liquid nitrogen

  • Consider perfusion fixation for animal tissues to improve antigen preservation

• Antigen retrieval techniques:

  • Implement heat-induced epitope retrieval using basic pH buffers (pH 9.0)

  • Optimize retrieval time (typically 15-20 minutes) to maximize antigen exposure without tissue damage

  • Allow slides to cool slowly in retrieval solution to prevent tissue detachment

• Section thickness optimization:

  • Use 5-8 μm sections for optimal antibody penetration and visualization

  • Ensure consistent section thickness across samples for comparative studies

• Blocking procedures:

  • Block with 5-10% normal serum (matching the species of the secondary antibody)

  • Include protein blockers (e.g., BSA) and detergents (e.g., 0.1-0.3% Triton X-100) to reduce background

  • Consider avidin/biotin blocking for protocols using biotin-based detection systems

Search results demonstrate successful MYH2 detection in both paraffin-embedded and frozen sections of human and mouse skeletal muscle using specific antibodies. For example, one protocol used heat-induced epitope retrieval with Antigen Retrieval Reagent-Basic followed by detection with Anti-Mouse IgG VisUCyte HRP Polymer Antibody and DAB staining, with hematoxylin counterstaining .

How can researchers accurately quantify MYH2 expression in mixed muscle samples?

Quantifying MYH2 expression in heterogeneous muscle samples requires sophisticated approaches:

• Image analysis for immunohistochemistry/immunofluorescence:

  • Utilize automated image analysis software to quantify MYH2-positive fibers

  • Calculate the percentage area of MYH2 staining relative to total muscle cross-section

  • Implement machine learning algorithms to classify fiber types in complex muscle compositions

  • Measure signal intensity as a semi-quantitative indicator of expression level

• Biochemical quantification methods:

  • Employ ELISA assays with a detection range of 0.156-10 ng/mL for precise MYH2 quantification

  • Use competitive ELISA formats when analyzing samples with potential interfering substances

  • Prepare standard curves with recombinant MYH2 protein for accurate concentration determination

• Normalization strategies:

  • Normalize Western blot data to total protein loading rather than single housekeeping proteins

  • Use ratios of MYH2 to other myosin isoforms to assess fiber type shifts

  • Include internal calibration standards in each experimental batch

• Single-cell approaches:

  • Implement laser capture microdissection to isolate specific fiber types before analysis

  • Consider single-cell RNA sequencing to correlate MYH2 mRNA with protein expression

When using ELISA methods, researchers should follow specific protocol steps, including proper reagent preparation (e.g., reconstituting standards with sample diluent to create a 10.0 ng/mL stock solution) and ensuring appropriate dilution of detection reagents as outlined in the search results .

How are MYH2 antibodies being utilized in studies of muscle regeneration and repair?

MYH2 antibodies provide valuable insights into muscle regeneration processes through several methodological approaches:

• Temporal expression analysis:

  • Track MYH2 expression during different phases of muscle regeneration

  • Correlate MYH2 appearance with satellite cell activation and fusion events

  • Monitor transitions between developmental and adult myosin isoforms during repair

• Therapeutic intervention assessment:

  • Evaluate how pharmacological or genetic interventions affect the restoration of normal MYH2 expression patterns

  • Compare regeneration efficiency by quantifying MYH2-positive fibers in treated versus control tissues

• Signaling pathway investigation:

  • Combine MYH2 staining with analysis of regeneration-associated signaling molecules

  • Assess how manipulation of pathways like Wnt/β-catenin affects MYH2 expression during repair

• Co-culture experimental systems:

  • Utilize MYH2 antibodies to evaluate the influence of non-muscle cells (e.g., macrophages, fibroblasts) on myotube formation and maturation

  • Quantify MYH2 expression as an indicator of functional recovery in co-culture models

The search results indicate that MYH2 expression can be modulated by various factors. For example, researchers discovered that knockdown of BAMBI inhibited myogenic differentiation as evidenced by reduced MYH2 expression, while LiCl treatment (which activates Wnt signaling) rescued this inhibitory effect . Similarly, miR-106a-5p was found to inhibit myogenic differentiation of C2C12 myoblasts, resulting in decreased MYH2 expression and affecting the PI3K-AKT pathway .

What considerations are important when using MYH2 antibodies in multi-species comparative studies?

When conducting comparative studies across species, researchers must consider several methodological factors:

• Cross-species reactivity verification:

  • Confirm antibody specificity for MYH2 in each species under investigation

  • Validate using positive control tissues from each species

  • Consider sequence homology analysis to predict potential cross-reactivity

• Epitope conservation analysis:

  • Select antibodies targeting highly conserved regions of MYH2 across species

  • Be aware that antibodies raised against human MYH2 may have varying affinity for orthologs in other species

  • Utilize bioinformatic tools to compare epitope sequences across target species

• Protocol optimization by species:

  • Adjust fixation and antigen retrieval conditions for each species' tissue characteristics

  • Modify antibody concentration and incubation times based on species-specific binding kinetics

  • Optimize blocking reagents to address species-specific background issues

• Data interpretation considerations:

  • Account for species differences in muscle fiber type distribution when comparing MYH2 expression

  • Recognize that fiber type nomenclature and myosin isoform composition may vary across species

  • Consider evolutionary context when interpreting cross-species differences

The search results indicate that MYH2 orthologs have been documented in mouse, rat, bovine, and chimpanzee species , and certain antibodies demonstrate cross-reactivity with human, mouse, rabbit, and rat MYH2 . For bovine samples specifically, an ELISA kit with a detection range of 0.156-10 ng/mL and minimum detection limit of 0.156 ng/mL is available .