MYOM2 Antibody

What is MYOM2 and why is it significant in muscle research?

MYOM2 (Myomesin-2) is a 165 kDa protein containing 1,465 amino acids that serves as a major component of the vertebrate myofibrillar M-band. The protein features a unique N-terminal domain followed by 12 repeat domains with strong homology to either fibronectin type III or immunoglobulin C2 domains. MYOM2 plays a critical mechanical role in muscle elasticity and contractility by linking myosin thick filaments to other structural proteins within the sarcomere. Recent research has identified MYOM2 mutations in patients with Tetralogy of Fallot (TOF) and hypertrophic cardiomyopathy (HCM), highlighting its significance in cardiac research . The protein's strategic location in the M-band makes it a valuable marker for studying sarcomere organization and muscle physiology.

How should I select the appropriate MYOM2 antibody for my specific research application?

Selection of an appropriate MYOM2 antibody depends on several experimental factors:

• Application compatibility: Verify the antibody has been validated for your intended application (WB, IHC, ICC, IP). For example, antibody CAB20526 has been validated for WB and ELISA applications , while antibody CAU21928 has been validated for WB, IHC, ICC, and IP .

• Species reactivity: Confirm cross-reactivity with your experimental model. Some antibodies like PA5-50064 react with human samples , while others like CAB20526 have broader reactivity across human, mouse, and rat models .

• Epitope recognition: Consider which domain of MYOM2 you need to target. Different antibodies recognize different regions - for example, ab233263 targets amino acids 1100-1450 , while CAU21928 targets Pro1130~Ser1434 .

• Validation evidence: Review available validation data. Prioritize antibodies with published validation images for your specific application.

What are the optimal fixation and tissue preparation methods for MYOM2 immunohistochemistry?

For optimal MYOM2 detection in immunohistochemistry, consider these methodological approaches:

• Fixation: Paraformaldehyde (PFA) fixation is recommended over other methods as it offers superior tissue penetration while preserving MYOM2 epitope integrity. PFA should be prepared fresh before use, as long-term stored PFA can polymerize into formalin, which may adversely affect antigen detection .

• Antigen retrieval: Heat-induced epitope retrieval in citrate buffer (pH 6.0) has been successfully employed with antibodies like ab233263, which has been validated for formalin-fixed, paraffin-embedded tissues .

• Blocking: A 5% BSA solution in PBS for 1 hour at room temperature helps reduce non-specific binding.

• Antibody concentration: For paraffin sections, a starting concentration of 20 μg/mL has shown successful staining with ab233263 , though optimization is recommended for each experimental system.

• Detection system: DAB (3,3'-diaminobenzidine) staining provides good visualization of MYOM2 in muscle and cardiac tissues, with minimal background when properly optimized .

What are the critical parameters for successful MYOM2 western blotting?

Successful western blotting for MYOM2 requires attention to several critical parameters:

• Sample preparation: Due to MYOM2's high molecular weight (165 kDa), use low percentage gels (6-8%) or gradient gels (4-15%) to ensure proper resolution.

• Transfer conditions: Extended transfer times (overnight at low voltage or 2-3 hours at higher voltage) are recommended for complete transfer of high molecular weight MYOM2.

• Antibody dilutions: Most MYOM2 antibodies work effectively at dilutions between 1:500 and 1:2000. For example, CAB20526 is recommended at 1:500-1:1000 , while others like ab233263 have been validated at 2 μg/mL .

• Positive controls: Heart and skeletal muscle lysates are ideal positive controls, as demonstrated in validation studies with antibodies like A12094, which shows clear detection in these tissues .

• Loading control selection: Standard loading controls like GAPDH (37 kDa) run at significantly different molecular weights than MYOM2 (165 kDa); consider using larger proteins like myosin heavy chain as alternative loading controls for normalization.

How can MYOM2 antibodies be used to investigate sarcomere disorganization in cardiomyopathies?

MYOM2 antibodies provide valuable tools for investigating sarcomere disorganization in cardiomyopathies through multiple methodological approaches:

• Comparative immunohistochemistry: Patient-derived cardiomyocytes from HCM patients with MYOM2 mutations exhibit myofibrillar disarray that can be visualized using MYOM2 antibodies alongside other sarcomeric markers. Quantitative analysis of sarcomere organization patterns between control and disease samples reveals structural abnormalities .

• Co-immunoprecipitation studies: MYOM2 antibodies can be used to investigate altered protein-protein interactions within the sarcomere. Research has shown that MYOM2 mutations may affect interactions with titin and myosin, potentially explaining the reduced passive force observed in patient-derived cardiomyocytes .

• Protein expression analysis: Western blotting with MYOM2 antibodies has revealed altered stoichiometry of sarcomeric proteins in HCM patients. For instance, analysis of HCM-02 myocardium showed reduced levels of cardiac troponin I (cTnI) and regulatory myosin light chain (MLC2v), with increased cardiac myosin-binding protein C (cMyBPC) .

• Phosphorylation studies: Combining MYOM2 antibodies with phospho-specific antibodies can reveal altered post-translational modifications. Patient samples carrying MYOM2 mutations showed reduced cTnI phosphorylation, suggesting downstream effects on regulatory mechanisms .

What strategies can resolve inconsistent MYOM2 antibody performance across different tissue types?

Researchers may encounter variable MYOM2 antibody performance across different tissue types. These methodological strategies can help address such inconsistencies:

• Epitope masking assessment: MYOM2's interactions with other sarcomeric proteins may mask epitopes in certain tissue contexts. Testing multiple antibodies targeting different regions of MYOM2 can help identify the most reliable detection method for each tissue type.

• Tissue-specific optimization protocols:

  Tissue Type	Recommended Fixation	Antigen Retrieval	Antibody Dilution
  Heart	4% PFA, 24h	Citrate buffer pH 6.0, 20 min	1:500
  Skeletal Muscle	4% PFA, 24h	EDTA buffer pH 9.0, 30 min	1:1000
  Brain	4% PFA, 48h	Tris-EDTA pH 9.0, 30 min	1:250

• Cross-validation approaches: When inconsistent results occur, employ multiple detection methods (e.g., IHC and WB) on the same sample to determine if the issue is application-specific or tissue-specific.

• Blocking optimization: Different tissues may require different blocking solutions. For highly vascularized tissues like heart, a combination of 5% BSA with 2% normal serum from the secondary antibody host species may reduce background.

• Sample preparation considerations: Muscle tissue often requires special handling to preserve sarcomeric structures. Flash-freezing samples in isopentane cooled with liquid nitrogen before sectioning can better preserve MYOM2 epitopes in skeletal muscle.

How are MYOM2 antibodies advancing our understanding of cardiac disease mechanisms?

MYOM2 antibodies have enabled significant advances in understanding cardiac disease mechanisms:

• Identification of MYOM2 as a novel disease gene: Antibody-based studies have helped establish MYOM2 as a candidate gene in hypertrophic cardiomyopathy (HCM) and Tetralogy of Fallot (TOF). Research identified four HCM patients with rare probable disease-causing mutations in MYOM2 who had negative screening results in the known HCM disease genes (MYH7, MYBPC3, TNNT2, TNNI3, TPM1, MYL2, MYL3, ACTC1, TCAP, TNNC1, MYOZ2, and CSRP3) .

• Sarcomeric protein stoichiometry analysis: MYOM2 antibodies revealed altered sarcomeric protein stoichiometry in HCM patients with MYOM2 mutations, showing reduced levels of cTnI and MLC2v with increased cMyBPC . This suggests MYOM2 mutations may have secondary effects on other sarcomere proteins.

• Functional implications: Patient-derived cardiomyocytes from individuals with MYOM2 mutations exhibit myofibrillar disarray and reduced passive force with increasing sarcomere lengths, providing insights into the mechanical consequences of MYOM2 dysfunction .

• Mechanistic pathway analysis: Combined use of MYOM2 antibodies with other sarcomeric markers has helped identify MYOM2 as a hub gene within interactions of sarcomere genes, suggesting its central role in coordinating sarcomere function .

What emerging methodologies are enhancing MYOM2 detection in complex tissue samples?

Advanced methodologies are improving MYOM2 detection in complex tissue samples:

• Multiplexed immunofluorescence: Combining MYOM2 antibodies with other sarcomeric markers (titin, myosin, α-actinin) in multiplexed imaging allows precise localization within the sarcomere structure and better visualization of structural abnormalities.

• Super-resolution microscopy: Techniques like STORM (Stochastic Optical Reconstruction Microscopy) and STED (Stimulated Emission Depletion) microscopy with appropriate MYOM2 antibodies enable nanoscale visualization of M-band organization beyond conventional confocal limits.

• Proximity ligation assays (PLA): This technique allows visualization of protein-protein interactions involving MYOM2 within the native tissue context, revealing how mutations affect MYOM2's interactions with binding partners like titin and myosin.

• Mass spectrometry-based validation: Using targeted proteomics to validate antibody specificity has emerged as a gold standard for confirming MYOM2 detection in complex samples, particularly when examining tissues with low expression levels.

• Cell-type specific analyses: Combining MYOM2 antibodies with cell-type specific markers enables distinction between MYOM2 expression patterns in different muscle fiber types or between cardiomyocyte subtypes within heterogeneous tissues.

What are the potential applications of MYOM2 antibodies in precision medicine approaches for cardiomyopathies?

MYOM2 antibodies hold promising applications for precision medicine in cardiomyopathies:

• Biomarker development: MYOM2 antibodies could help develop diagnostic assays to detect circulating MYOM2 fragments in patients with sarcomeric dysfunction, potentially serving as early biomarkers of cardiomyopathy progression.

• Patient stratification: Immunohistochemical analysis using MYOM2 antibodies on myocardial biopsies might help classify HCM patients into molecular subtypes, potentially identifying those with primary sarcomeric M-band abnormalities versus other mechanisms.

• Therapeutic response monitoring: MYOM2 antibodies could monitor sarcomeric reorganization in response to emerging therapies targeting sarcomere function, providing structural endpoints beyond functional assessments.

• Personalized therapeutic approaches: Understanding the specific molecular consequences of different MYOM2 mutations through antibody-based studies may guide selection of personalized therapeutic strategies targeting the downstream pathways affected.

• Genetic variant classification: Antibody-based functional studies can help classify variants of uncertain significance in MYOM2, providing evidence for or against pathogenicity in cases where genetic information alone is insufficient.

How might comparative species analysis with MYOM2 antibodies inform evolutionary perspectives on sarcomere structure?

Comparative species analysis using MYOM2 antibodies offers valuable evolutionary insights:

• Conservation analysis: MYOM2 antibodies with cross-reactivity across species (human, mouse, rat, pig, and others) enable direct comparison of M-band organization across evolutionary distances, revealing conserved versus divergent features .

• Functional adaptation studies: Comparing MYOM2 expression and localization between species with different cardiac or skeletal muscle physiological demands (e.g., comparing human heart to mouse or comparing slow-twitch vs. fast-twitch muscles across species) could reveal adaptive specializations.

• Ortholog identification: Research suggests that the Drosophila gene CG14964 (referred to as dMnM) may be an ortholog of MYOM2 . Cross-species antibody studies could help confirm functional equivalence and evolutionary relationships.

• Developmental pattern analysis: MYOM2 antibodies can reveal differences in sarcomere assembly timing and patterns across species during embryonic development, providing insights into evolutionary changes in muscle development programs.

• Disease model validation: Comparative immunostaining with MYOM2 antibodies across species helps validate animal models of human cardiac disease, ensuring the same structural abnormalities are present despite species differences.