MYOZ2 Antibody, Biotin conjugated

What is MYOZ2 and what are its functional roles in cellular physiology?

MYOZ2 (Myozenin-2) is a 30 kDa protein encoded by the MYOZ2 gene that belongs to the myozenin family of sarcomeric proteins. It primarily localizes to the Z-line of the sarcomere in cardiac and skeletal muscle cells where it colocalizes with alpha-actinin (ACTN1) and calcineurin (PPP3CA) . MYOZ2 functions as a calcineurin-binding protein, tethering this phosphatase to alpha-actinin at the Z-line, thereby playing a crucial role in calcium-dependent signal transduction pathways in muscle cells .

Mutations in the MYOZ2 gene are associated with familial hypertrophic cardiomyopathy type 16 (CMH16), a hereditary heart disorder characterized by ventricular hypertrophy . This condition exhibits variable expressivity, ranging from benign to malignant forms with high risk of cardiac failure and sudden cardiac death . Recent research has also uncovered an unexpected role for MYOZ2 in adipogenesis, with evidence suggesting that MYOZ2 expression patterns change during adipogenic differentiation .

How do I select the appropriate MYOZ2 antibody for my specific research application?

When selecting a MYOZ2 antibody for your research, consider the following critical factors:

• Target species reactivity: MYOZ2 antibodies show varying reactivity profiles. For example, antibody 11450-1-AP demonstrates reactivity with human, mouse, and rat samples , while E-AB-52584 has confirmed reactivity with human and mouse samples .

• Validated applications: Match the antibody to your intended application. The table below summarizes validated applications for common MYOZ2 antibodies:

• Clone type: Consider whether a polyclonal or monoclonal antibody better suits your needs. Both antibodies in the search results are polyclonal, which may provide broader epitope recognition but potentially less specificity than monoclonal alternatives .

• Conjugation status: For direct detection methods, conjugated antibodies (e.g., biotin-conjugated) eliminate the need for secondary antibodies. For traditional detection methods, unconjugated antibodies like 11450-1-AP and E-AB-52584 are suitable .

What are the characteristics and molecular properties of MYOZ2 protein that affect antibody selection?

MYOZ2 has several characteristics that should inform antibody selection:

• Molecular weight considerations: While the calculated molecular weight of MYOZ2 is 30 kDa, the observed molecular weight in experimental conditions is typically 30-35 kDa . This discrepancy may be due to post-translational modifications or other factors affecting protein mobility during electrophoresis.

• Localization patterns: MYOZ2 localizes specifically to the Z-line of the sarcomere in cardiac and skeletal muscle tissues . For accurate detection in immunohistochemistry or immunofluorescence applications, antibodies must maintain specificity for this subcellular localization.

• Tissue expression profile: MYOZ2 is predominantly expressed in heart and skeletal muscle tissues . When selecting antibodies for other tissue types, validation is crucial as expression levels may be significantly lower.

• Protein synonyms and database identifiers: MYOZ2 is known by multiple names (Calsarcin-1, CS-1, FATZ-related protein 2) . When searching databases, use all relevant identifiers including gene ID (51778) and UniProt ID (Q9NPC6) to ensure comprehensive results.

How can I optimize Western blot protocols for reliable MYOZ2 detection?

Optimizing Western blot protocols for MYOZ2 detection requires attention to several factors:

• Sample preparation: For MYOZ2 detection, heart and skeletal muscle tissues have been validated as reliable sources . When extracting protein, use buffers containing protease inhibitors to prevent degradation of the target protein.

• Antibody dilution optimization: Different MYOZ2 antibodies require different dilutions. For example:

  • For 11450-1-AP: Use 1:2000-1:12000 dilution for WB applications

  • For E-AB-52584: Use 1:500-1:2000 dilution for WB applications

• Addressing molecular weight discrepancies: As noted in the Elabscience documentation, "The actual band is not consistent with the expectation." The mobility of proteins during electrophoresis can be affected by many factors, including post-translational modifications . When detecting MYOZ2, expect bands around 30-35 kDa rather than exactly at the calculated 30 kDa size .

• Detection method considerations: For enhanced sensitivity when using biotin-conjugated antibodies, consider using streptavidin-HRP systems with appropriate blocking solutions to minimize background.

• Buffer conditions: Store MYOZ2 antibodies according to manufacturer recommendations. For instance, the 11450-1-AP antibody should be stored in PBS with 0.02% sodium azide and 50% glycerol at pH 7.3 .

What methodological challenges exist when studying MYOZ2 in different disease models?

Studying MYOZ2 in disease models presents several methodological challenges:

• Familial hypertrophic cardiomyopathy (CMH16) models: When investigating MYOZ2 mutations associated with CMH16, researchers must account for the inter- and intrafamilial variability of the disorder . This heterogeneity necessitates careful phenotyping and correlation between MYOZ2 expression/mutation status and disease severity.

• Epigenetic considerations: Recent research indicates that DNA methylation affects MYOZ2 expression. The promoter region of MYOZ2 contains multiple methylation sites that can influence gene expression . This epigenetic regulation adds complexity when interpreting MYOZ2 expression data in disease models.

• Interaction with lncRNA: The long non-coding RNA lncMYOZ2 has been shown to influence MYOZ2 expression through an AHCY/DNMT1-dependent mechanism affecting DNA methylation of the MYOZ2 promoter . This regulatory relationship should be considered when designing experiments to study MYOZ2 function.

• Adipogenesis models: Emerging evidence suggests MYOZ2 plays a role in adipogenesis, with expression patterns changing during adipogenic differentiation . When studying this context, researchers should note that MYOZ2 was "downregulated 2 d after adipogenic induction but then increased gradually during the course of adipogenesis" .

How does MYOZ2 interact with other proteins in the Z-line complex, and how can these interactions be studied?

MYOZ2 forms part of a complex protein network at the Z-line of the sarcomere:

• Key interaction partners: MYOZ2 interacts with:

  • Alpha-actinin (ACTN1) - a structural component of the Z-line

  • Calcineurin (PPP3CA) - a phosphatase involved in calcium-dependent signal transduction

  • Telethonin - as indicated by GO annotations related to telethonin binding

• Methodological approaches for studying interactions:

  • Immunoprecipitation (IP) followed by Western blotting can confirm direct protein-protein interactions

  • Proximity ligation assays (PLA) can visualize protein interactions in situ

  • For studying interactions in the context of the Z-line structure, super-resolution microscopy provides the necessary spatial resolution

• Functional significance: The interaction between MYOZ2 and calcineurin is particularly important as it positions this phosphatase at the Z-line, influencing calcium-dependent signaling pathways in muscle cells . Disruption of this interaction may contribute to the pathophysiology of cardiomyopathies.

How can I optimize immunohistochemistry protocols for MYOZ2 detection in different tissue types?

Optimizing immunohistochemistry for MYOZ2 detection requires tissue-specific considerations:

• Antigen retrieval methods: For the 11450-1-AP antibody, different antigen retrieval methods are recommended depending on the tissue:

  • Suggested primary method: TE buffer pH 9.0

  • Alternative method: Citrate buffer pH 6.0

• Tissue-specific dilutions: While the 11450-1-AP antibody has a recommended dilution range of 1:50-1:500 for IHC applications , optimal dilutions may vary by tissue type. The manufacturer notes that dilutions are "sample-dependent" and recommends titrating the reagent in each testing system .

• Validated tissue types: The 11450-1-AP antibody has been validated for:

  • Human heart tissue

  • Mouse brain tissue

For other tissue types, additional validation steps are necessary.

• Controls: Include both positive controls (tissues known to express MYOZ2, such as heart or skeletal muscle) and negative controls (tissues with low or no MYOZ2 expression) in your experimental design.

What are the best approaches for validating MYOZ2 antibody specificity for critical research applications?

Validating MYOZ2 antibody specificity is essential for reliable research outcomes:

• Knockout/knockdown validation: Use MYOZ2 knockdown samples as negative controls. Research has demonstrated successful MYOZ2 knockdown using siRNA transfection in porcine preadipocytes, with confirmation of reduced MYOZ2 at both mRNA and protein levels .

• Multiple antibody approach: Compare results from different MYOZ2 antibodies targeting distinct epitopes. The search results provide information on two different antibodies (11450-1-AP and E-AB-52584), which could be used comparatively .

• Peptide competition assays: Pre-incubate the antibody with the immunizing peptide or recombinant MYOZ2 protein before application to confirm binding specificity.

• Molecular weight confirmation: Verify that detected bands correspond to the expected molecular weight range for MYOZ2 (30-35 kDa) , keeping in mind that mobility can be affected by post-translational modifications.

• Cross-reactivity assessment: As MYOZ2 belongs to a family of myozenin proteins, assess potential cross-reactivity with paralogous proteins like MYOZ1, which is noted as an important paralog of MYOZ2 .

How can I troubleshoot inconsistent results when using MYOZ2 antibodies in complex experimental systems?

When troubleshooting inconsistent results with MYOZ2 antibodies:

• Inconsistent molecular weight detection: As noted in the Elabscience documentation, "The mobility is affected by many factors, which may cause the observed band size to be inconsistent with the expected size." The mobility can be influenced by:

  • Post-translational modifications

  • Different modified forms of the protein present simultaneously

  • Sample preparation methods affecting protein denaturation

• Antibody storage and handling: Follow manufacturer recommendations for storage conditions:

  • 11450-1-AP: Store at -20°C. Stable for one year after shipment. Aliquoting is unnecessary for -20°C storage

  • E-AB-52584: Store at -20°C. Valid for 12 months. Avoid freeze/thaw cycles

• Sample preparation optimization: Ensure complete lysis and denaturation of proteins from Z-line structures, which may require specialized extraction protocols for muscle tissue.

• Antibody lot variation: Document the lot number of antibodies used and consider testing multiple lots if inconsistent results are observed between experiments.

• Signal amplification strategies: For low abundance detection, consider biotin-conjugated secondary antibodies or biotin-conjugated primary antibodies with streptavidin amplification systems to enhance detection sensitivity.

How is MYOZ2 research contributing to our understanding of cardiomyopathies and potential therapeutic targets?

MYOZ2 research has significant implications for understanding cardiomyopathies:

• Genetic basis of familial hypertrophic cardiomyopathy: Defects in the MYOZ2 gene cause familial hypertrophic cardiomyopathy type 16 (CMH16), characterized by ventricular hypertrophy . Understanding the specific mutations and their functional consequences provides insight into disease mechanisms.

• Variability in disease presentation: CMH16 exhibits significant inter- and intrafamilial variability, ranging from benign forms to malignant forms with high risk of cardiac failure and sudden cardiac death . MYOZ2 antibodies are essential tools for studying how variations in MYOZ2 expression correlate with this clinical heterogeneity.

• Calcineurin signaling in heart disease: As MYOZ2 tethers calcineurin to alpha-actinin at the Z-line , dysregulation of this interaction may affect calcium-dependent signaling pathways implicated in cardiac hypertrophy. This positioning makes MYOZ2 a potential modulator of calcineurin activity in heart muscle.

• Variants of unknown significance (VUS): Recent research has highlighted the challenge of interpreting variants of unknown significance in inherited arrhythmic syndromes, including those affecting MYOZ2 . Antibody-based functional studies can help classify these variants and improve genetic diagnosis.

What is the emerging role of MYOZ2 in adipogenesis and metabolic research?

Recent research has uncovered unexpected roles for MYOZ2 in adipose tissue:

• Expression pattern during adipogenesis: MYOZ2 expression is dynamically regulated during adipogenic differentiation, being "downregulated 2 d after adipogenic induction but then increased gradually during the course of adipogenesis" . This pattern suggests a regulatory role in adipocyte development.

• Functional requirement in adipocyte formation: Knockdown of MYOZ2 in porcine preadipocytes resulted in "a drastic decrease in lipid accumulation" and reduced expression of adipocyte markers CEBPα, PPARγ, and FABP4 at both mRNA and protein levels . These findings establish "an essential role for MYOZ2 in regulating adipogenesis in pigs" .

• Epigenetic regulation through lncRNA: The long non-coding RNA lncMYOZ2 influences MYOZ2 expression through an epigenetic mechanism involving AHCY/DNMT1-dependent regulation of DNA methylation at the MYOZ2 promoter . This regulatory network adds complexity to MYOZ2 function in adipose tissue.

• Implications for metabolic research: Given the critical role of adipogenesis in metabolic disorders, MYOZ2 may represent a novel factor in obesity and related conditions. MYOZ2 antibodies will be crucial tools for investigating this protein's distribution and abundance in adipose tissue from different metabolic states.

How can biotin-conjugated MYOZ2 antibodies enhance multiplex immunoassay development for complex tissue analysis?

Biotin-conjugated MYOZ2 antibodies offer several advantages for multiplex immunoassays:

• Amplification of detection signals: The high-affinity interaction between biotin and streptavidin provides signal amplification, enhancing detection sensitivity in tissues with low MYOZ2 expression.

• Compatibility with multiple detection systems: Biotin conjugation enables flexible experimental design through compatibility with various streptavidin-conjugated reporters (HRP, fluorophores, quantum dots) without changing the primary antibody.

• Multiplexing capabilities: In complex tissue analyses requiring simultaneous detection of multiple proteins, biotin-conjugated MYOZ2 antibodies can be paired with antibodies using different conjugation systems to achieve distinctive labeling.

• Application in detecting MYOZ2 in non-muscle tissues: The signal amplification provided by biotin-streptavidin systems may be particularly valuable when investigating MYOZ2 in tissues where it is expressed at lower levels than in cardiac and skeletal muscle.