MYOM1 Antibody

What is MYOM1 and why is it important in structural biology research?

MYOM1 (Myomesin-1) is a major component of the vertebrate myofibrillar M-band that functions as a structural protein connecting myosin and titin in sarcomeres. It binds myosin, titin, and light meromyosin in a dose-dependent manner . As a 188-190 kDa protein, MYOM1 is critical for maintaining sarcomere integrity, making it an important target for muscle biology research. The protein contains structural modules with strong homology to fibronectin type III and immunoglobulin C2 domains arranged in a specific pattern (II-II-I-I-I-I-I-II-II-II-II-II) . Studying MYOM1 is particularly valuable for understanding the molecular architecture of muscle fibers and potentially elucidating mechanisms underlying myopathies and cardiac disorders.

What types of MYOM1 antibodies are available for research applications?

MYOM1 antibodies are available in several formats based on host species, clonality, and production methods:

When selecting an antibody, researchers should consider the specific experimental requirements, including the technique being employed, species cross-reactivity needs, and the required sensitivity level. Recombinant monoclonal antibodies offer the advantage of renewable supply with consistent performance .

What are the optimal dilutions and experimental conditions for MYOM1 antibody applications?

Optimal working dilutions vary significantly depending on the application and specific antibody:

For Western blotting, many MYOM1 antibodies show optimal results at higher dilutions (1:20,000) when using heart or skeletal muscle tissue lysates at 20 μg protein loading . For challenging samples, optimization by titration is strongly recommended.

Which tissue samples provide the strongest MYOM1 signal for antibody validation?

Based on expression patterns, the following tissues are optimal for MYOM1 detection:

Mouse and rat heart tissues consistently show strong expression and are recommended for initial antibody validation experiments . Human skeletal muscle also provides robust signals for immunohistochemical applications .

How can I preserve MYOM1 antibody activity during storage and handling?

Proper storage is critical for maintaining antibody functionality:

• For short-term use (≤2 weeks), store at 4°C

• For long-term storage, aliquot in volumes ≥20 μL and store at -20°C or -80°C to avoid freeze-thaw cycles

• For concentrates, consider adding equal volume of glycerol as a cryoprotectant before freezing

• Many MYOM1 antibodies are supplied in PBS with 0.02% sodium azide and 50% glycerol at pH 7.3-7.4

Researchers should minimize freeze-thaw cycles, as repeated freezing and thawing can significantly reduce antibody activity and specificity. Proper aliquoting upon first thaw is strongly recommended.

What are the most effective methods for validating MYOM1 antibody specificity?

Multiple orthogonal approaches should be employed to ensure antibody specificity:

• Western blot with positive/negative controls: Compare heart/skeletal muscle (positive) against tissues with minimal expression like kidney or brain

• Knockdown/knockout validation: Use siRNA or CRISPR techniques targeting MYOM1 to demonstrate signal reduction

• Immunoprecipitation coupled with mass spectrometry (IP-MS): This approach can identify the target protein, interacting partners, and potential off-targets with high confidence9

• Orthogonal antibody testing: Compare results using antibodies targeting different epitopes of MYOM1

• RNAseq correlation: Compare protein expression with transcript levels across tissues

A comprehensive validation strategy employing multiple techniques is considered the gold standard. The independent validation approach using orthogonal methods provides the strongest evidence for antibody specificity 9.

How can researchers address potential cross-reactivity issues with MYOM1 antibodies?

Cross-reactivity concerns can be mitigated through several approaches:

• Epitope analysis: Select antibodies targeting unique regions of MYOM1 that have minimal sequence homology with related proteins like MYOM2

• Blocking peptide experiments: Pre-incubate antibody with immunizing peptide to confirm signal specificity

• Multiple antibody validation: Use at least two antibodies targeting different epitopes to confirm localization patterns

• Careful selection of negative controls: Include tissues known to express low levels of MYOM1 but potentially high levels of homologous proteins

• Species considerations: When testing in non-human models, evaluate sequence homology in the epitope region

The main concerns for cross-reactivity are with other members of the myomesin family, particularly MYOM2, which shares approximately 50% sequence identity in the repeat-containing region .

What technical challenges should be anticipated when studying MYOM1 in muscle samples?

Working with muscle tissue presents specific challenges:

• High protein content: Muscle lysates often require higher dilutions of primary antibody (1:20,000-1:100,000) to prevent background

• Tissue processing: Proper fixation is critical - overfixation can mask epitopes in sarcomeric proteins

• Antigen retrieval optimization: Different epitopes may require specific retrieval conditions; testing both citrate buffer (pH 6.0) and TE buffer (pH 9.0) is recommended

• Sample preparation for Western blotting: Efficient extraction of sarcomeric proteins requires specialized lysis buffers that can solubilize structural proteins

• Molecular weight verification: The observed molecular weight can vary from the predicted 188 kDa depending on tissue source and sample preparation

When working with cardiac or skeletal muscle tissues, researchers should optimize protein extraction protocols specifically for sarcomeric proteins, which can be challenging to solubilize completely.

How should experimental design be adapted for studying MYOM1 interactions with other sarcomeric proteins?

To effectively study protein-protein interactions involving MYOM1:

• Co-immunoprecipitation strategy:

  • Use antibodies validated for immunoprecipitation applications

  • Consider gentle lysis conditions to preserve protein complexes

  • Include appropriate controls to distinguish specific from non-specific interactions

• Proximity ligation assays (PLA):

  • Particularly useful for detecting MYOM1 interactions with titin and myosin in intact tissue

  • Requires antibodies raised in different host species

• Pull-down assays with recombinant domains:

  • Map interaction domains using recombinant fragments of MYOM1

  • Consider the modular structure of MYOM1 with its immunoglobulin and fibronectin domains

• In situ analysis:

  • Combine immunofluorescence with super-resolution microscopy to visualize co-localization at the M-band

  • Use dual-labeling techniques with established M-band markers

When designing interaction experiments, researchers should consider that MYOM1 binding to myosin, titin, and light meromyosin is dose-dependent , suggesting potential regulatory mechanisms worth investigating.

What methodological considerations are important when using MYOM1 antibodies for disease research?

When investigating MYOM1 in disease contexts:

• Sample selection and preparation:

  • For cardiac pathologies, consider spatial heterogeneity within diseased hearts

  • For biopsies, ensure adequate sampling from representative regions

  • Standardize fixation protocols across all comparison groups

• Quantitative analysis approaches:

  • Normalize MYOM1 expression to appropriate loading controls (tissue-specific considerations)

  • For densitometry, use multiple exposure times to ensure linearity of signal

  • Consider using multiple antibodies to confirm expression changes

• Interpretation challenges:

  • Distinguish between primary alterations in MYOM1 and secondary changes due to sarcomere remodeling

  • Consider post-translational modifications that may affect antibody recognition

  • Evaluate transcript levels alongside protein expression when possible

• Technical controls for disease studies:

  • Include non-affected tissue regions from the same sample when possible

  • Age-match controls carefully for developmental studies

  • Consider using both Western blot and immunohistochemistry to distinguish expression changes from localization changes

Researchers should be particularly careful when interpreting changes in MYOM1 levels or localization in disease states, as altered antibody accessibility or epitope masking could occur in pathological sarcomeric structures.

How can MYOM1 antibodies be effectively employed in chromatin immunoprecipitation (ChIP) experiments?

Although MYOM1 is not typically a focus of ChIP studies as it's not a transcription factor, the principle of immunoprecipitation optimization applies:

• Antibody selection:

  • For transcription factor studies that might regulate MYOM1, such as androgen receptor (AR) which has been shown to modulate expression of sarcomeric genes

  • Choose antibodies specifically validated for ChIP applications

• Protocol optimization:

  • When studying factors binding to the MYOM1 gene, use appropriate crosslinking conditions

  • Consider sonication parameters carefully to generate optimal fragment sizes

  • Include negative control regions and IgG controls

• Data analysis considerations:

  • Normalize ChIP-qPCR data appropriately for primer efficiency

  • For genome-wide studies, include validation of selected binding regions by ChIP-qPCR

  • Consider biological replicates to account for variability

When studying transcription factor binding to the MYOM1 gene, researchers have successfully used ChIP-qPCR to validate binding events originally identified in ChIP-on-chip experiments , demonstrating the importance of orthogonal validation in chromatin studies.

What advanced imaging techniques are most suitable for MYOM1 localization studies?

For precise localization of MYOM1 within the sarcomere:

• Super-resolution microscopy approaches:

  • STED (Stimulated Emission Depletion) microscopy provides resolution beyond the diffraction limit

  • STORM/PALM techniques offer single-molecule localization precision

  • SIM (Structured Illumination Microscopy) provides improved resolution for co-localization studies

• Sample preparation considerations:

  • Thin sectioning (5-10 μm) for muscle tissue is critical

  • For cultured cardiomyocytes, optimal fixation to preserve sarcomere structure is essential

  • Appropriate blocking to reduce non-specific binding in muscle tissue

• Multi-labeling strategies:

  • Combine MYOM1 staining with other M-band markers

  • Use Z-disc markers (e.g., α-actinin) as reference points for sarcomere organization

  • Consider triple-labeling with titin antibodies recognizing different regions

• 3D reconstruction approaches:

  • Z-stack acquisition for complete sarcomere visualization

  • Deconvolution processing to improve signal-to-noise ratio

  • Volume rendering for comprehensive structural analysis