MYOZ2 Antibody, HRP conjugated

What is MYOZ2 and what is its biological significance?

MYOZ2 (Myozenin-2), also known as Calsarcin-1 or FATZ-related protein 2, is a sarcomeric protein that serves as an intracellular binding protein involved in linking Z-line proteins such as alpha-actinin, gamma-filamin, TCAP/telethonin, and LDB3/ZASP while localizing calcineurin signaling to the sarcomere . The protein plays a critical role in the modulation of calcineurin signaling and may be involved in myofibrillogenesis .

MYOZ2 is particularly significant in muscle biology because defects in the MYOZ2 gene are associated with familial hypertrophic cardiomyopathy type 16 (CMH16), a hereditary heart disorder characterized by ventricular hypertrophy . The disorder shows variable clinical presentation ranging from benign to malignant forms with high risk of cardiac failure and sudden cardiac death .

What are the key applications for MYOZ2 antibody with HRP conjugation?

MYOZ2 antibody with HRP conjugation is specifically optimized for the following applications:

*While HRP-conjugated antibodies are primarily used for ELISA and WB applications, they can sometimes be utilized in IHC with appropriate detection systems .

What tissue types and species reactivity should researchers consider when using MYOZ2 antibodies?

Based on validation data:

MYOZ2 shows high expression in cardiac and skeletal muscle tissues, with particularly strong signals in heart samples. When designing experiments, consider that MYOZ2 is primarily localized to the cytoplasm, specifically at the myofibril sarcomere Z-line where it colocalizes with ACTN1 and PPP3CA .

What are the critical parameters for optimizing Western blot protocols with HRP-conjugated MYOZ2 antibodies?

When optimizing Western blot protocols with HRP-conjugated MYOZ2 antibodies, consider the following:

• Sample preparation: For optimal results, extract proteins from heart tissue or skeletal muscle using buffers containing protease inhibitors to prevent degradation of MYOZ2 protein .

• Loading control selection: Given the tissue-specific expression of MYOZ2, traditional housekeeping proteins like GAPDH may not provide optimal normalization. Consider using muscle-specific loading controls such as sarcomeric actin .

• Expected molecular weight: MYOZ2 has a calculated molecular weight of 30 kDa, but the observed molecular weight typically ranges between 30-35 kDa . This difference may be due to post-translational modifications.

• Dilution optimization: Start with a 1:500 dilution for Western blot and adjust as needed. For unconjugated antibodies, dilutions from 1:2000 to 1:12000 have been reported effective, but HRP-conjugated versions typically require higher concentrations (1:100-500) .

• Membrane type: PVDF membranes have shown good results for MYOZ2 detection.

Note that the mobility of MYOZ2 in SDS-PAGE may be affected by various factors, potentially causing the observed band size to be inconsistent with expectations .

What antigen retrieval methods and controls are recommended for MYOZ2 immunohistochemistry?

For optimal immunohistochemical detection of MYOZ2:

Antigen Retrieval Methods:

• Primary recommendation: TE buffer at pH 9.0 has shown effective results for MYOZ2 epitope exposure .

• Alternative method: Citrate buffer at pH 6.0 can be used as an alternative approach if TE buffer proves suboptimal .

Control Recommendations:

• Positive control tissues: Use human heart tissue or mouse heart tissue where MYOZ2 is known to be abundantly expressed .

• Negative control: Include skeletal muscle from MYOZ2 knockout mice (if available) or tissues known to lack MYOZ2 expression.

• Antibody controls: Include a control slide with secondary antibody only to assess non-specific binding.

When evaluating IHC results, look for specific staining at the Z-lines of sarcomeres, as MYOZ2 colocalizes with α-actinin and calcineurin at these structures .

How can researchers verify the specificity of their MYOZ2 antibody?

To verify antibody specificity:

• Western blot validation: Confirm a predominant band at 30-35 kDa in heart and skeletal muscle lysates .

• Knockout validation: If available, test the antibody on samples from MYOZ2 knockout mice (Myoz1 KO or Myoz2 KO), which should show absence of specific signal .

• Competitive binding assay: Pre-incubate the antibody with recombinant MYOZ2 protein before application to target samples; this should diminish or eliminate specific binding.

• Co-localization studies: In immunofluorescence applications, confirm that MYOZ2 co-localizes with other known Z-line proteins such as α-actinin .

• Multiple antibody approach: Use antibodies targeting different epitopes of MYOZ2 to confirm staining patterns.

How can MYOZ2 antibodies be used to investigate calcineurin signaling in muscle tissue?

MYOZ2 plays a crucial role in modulating calcineurin signaling within the sarcomere. To investigate this relationship:

• Co-immunoprecipitation (Co-IP) studies: Use MYOZ2 antibodies to pull down protein complexes and analyze calcineurin interaction partners. Apply protocols similar to those used in previous studies that identified Z-disc interaction partners including α-actinin, gamma-filamin, and calcineurin .

• NFAT activity assays: Combined with MYOZ2 overexpression or knockdown, measure NFAT activity using reporter assays. Research has shown that calsarcin-2 (MYOZ1) inhibits calcineurin/NFAT signaling, with similar functions potentially attributed to MYOZ2 .

• Phosphorylation state analysis: Examine how MYOZ2 affects the phosphorylation state of calcineurin substrates such as NFAT transcription factors. Western blot analysis using phospho-specific antibodies can reveal changes in NFAT phosphorylation following MYOZ2 manipulation .

• RCAN1-4 expression analysis: Monitor RCAN1-4 (regulator of calcineurin 1-4) expression as a readout of calcineurin activity in the context of MYOZ2 modulation. Increased RCAN1-4 expression indicates enhanced calcineurin signaling .

• Muscle fiber type analysis: Assess how MYOZ2 manipulation affects the proportion of oxidative (type I/IIa) versus glycolytic (type IIb) muscle fibers, as calcineurin signaling influences fiber type determination .

What experimental approaches can link MYOZ2 to muscle differentiation and fiber type determination?

Research has shown that MEF2A regulates myoblast differentiation via MYOZ2 . To investigate this relationship:

• Knockdown/overexpression studies: Manipulate MYOZ2 expression in myoblast cultures using siRNA or expression vectors, then assess differentiation markers and myotube formation.

• Chromatin immunoprecipitation (ChIP): Investigate MEF2A binding to the MYOZ2 promoter region. Studies have shown that MEF2A acts as a transcription factor that binds to the MYOZ2 proximal promoter .

• Fiber type analysis techniques:

  • Immunohistochemistry: Use antibodies against myosin heavy chain isoforms to classify fiber types.

  • Succinate dehydrogenase (SDH) staining: Identify oxidative fibers, which have increased in MYOZ1 knockout mice studies .

  • ATPase staining: Differentiate between fast-twitch and slow-twitch fibers.

• Gene expression profiling: Analyze changes in expression of fiber-type specific genes following MYOZ2 manipulation, particularly those regulated by calcineurin/NFAT signaling.

• Exercise performance testing: In animal models, assess how MYOZ2 manipulation affects exercise capacity, as MYOZ1-deficient mice showed markedly improved performance and enhanced running distances .

How can researchers interpret inconsistent molecular weight observations when detecting MYOZ2?

When encountering variations in MYOZ2 molecular weight:

• Expected vs. observed weight: MYOZ2 has a calculated molecular weight of 30 kDa, but is typically observed at 30-35 kDa on Western blots .

• Post-translational modifications: Consider that MYOZ2 may undergo phosphorylation, ubiquitination, or other modifications that alter mobility.

• Tissue-specific variations: Different tissue types may express MYOZ2 with different modifications or isoforms.

• Multiple bands interpretation: If multiple bands are observed, consider:

  • Degradation products (lower molecular weight bands)

  • Alternative splice variants

  • Different post-translational modification states

As noted by Elabscience: "The mobility is affected by many factors, which may cause the observed band size to be inconsistent with the expected size. The common factors include: If a protein in a sample has different modified forms at the same time, multiple bands may be detected on the membrane."

How should researchers address weak or inconsistent signals when using HRP-conjugated MYOZ2 antibodies?

When experiencing signal issues:

• Sample quality assessment: Ensure protein integrity by adding fresh protease inhibitors during extraction and keeping samples cold throughout processing.

• Blocking optimization: Test different blocking agents (BSA vs. non-fat dry milk) as MYOZ2 detection may be sensitive to blocking conditions.

• Signal amplification: For weak signals, consider:

  • Extended exposure times for Western blots

  • Enhanced chemiluminescence (ECL) substrate optimized for HRP detection

  • Tyramide signal amplification (TSA) for immunohistochemistry

• Storage conditions review: HRP-conjugated antibodies should be stored at -20°C and are stable for one year after shipment . Avoid repeated freeze-thaw cycles which may compromise HRP activity.

• Buffer compatibility: Ensure buffers used are compatible with HRP activity; phosphate buffers at pH 7.3-7.4 are typically optimal .

What are the key considerations for interpreting MYOZ2 expression patterns across different muscle types?

When analyzing MYOZ2 expression:

• Fiber type composition awareness: MYOZ2 expression may vary with muscle fiber type composition. Type I (slow-twitch) and Type II (fast-twitch) fibers show different expression patterns of myozenin family members .

• Developmental stage consideration: Expression patterns may differ between developing and mature muscle. Investigate temporal expression profiles when studying developmental processes.

• Disease state impact: In conditions like hypertrophic cardiomyopathy, MYOZ2 expression or localization may be altered . Compare pathological samples with appropriate controls.

• Cross-reactivity assessment: Validate that your antibody distinguishes between MYOZ2 and other myozenin family members (MYOZ1 and MYOZ3), which share sequence homology but have distinct expression patterns .

• Quantification approach: When quantifying MYOZ2 levels:

  • Normalize to appropriate loading controls

  • Consider using multiple detection methods (Western blot and qPCR)

  • Correlate with functional parameters when possible

How can researchers optimize experimental design when studying MYOZ2 in the context of muscle disease models?

For studies involving muscle disease models:

• Disease-relevant models selection: Choose models that recapitulate key aspects of diseases associated with MYOZ2 dysfunction, such as hypertrophic cardiomyopathy .

• Temporal analysis planning: Design experiments to capture both acute and chronic changes in MYOZ2 expression and function.

• Combined methodological approach: Integrate:

  • Protein expression analysis (Western blot, immunohistochemistry)

  • mRNA expression analysis (qPCR, RNA-seq)

  • Functional assessments (muscle strength, exercise capacity)

  • Structural analysis (sarcomere organization, Z-line integrity)

• Genetic manipulation strategies: Consider:

  • CRISPR/Cas9-mediated gene editing

  • Transgenic overexpression of wild-type or mutant MYOZ2

  • Conditional knockout models to study tissue-specific effects

• Translational relevance enhancement: Include human samples when available to validate findings from animal models, as MYOZ2 antibodies have demonstrated reactivity with human tissues .

How does MYOZ2 interact with MEF2A in regulating myoblast differentiation?

Recent research has revealed a significant relationship between MEF2A and MYOZ2 in muscle development:

• Transcriptional regulation: MEF2A has been identified as a transcription factor that binds to the MYOZ2 proximal promoter and functions upstream of MYOZ2 during myoblast differentiation .

• Expression dynamics: During myogenesis of bovine skeletal muscle primary myoblast, the mRNA expression level of MEF2A dramatically increases, suggesting its importance in regulating differentiation-associated genes like MYOZ2 .

• Functional consequences: MEF2A promotes myoblast proliferation, while its knockdown inhibits both proliferation and differentiation. This effect is mediated in part through regulation of MYOZ2 expression .

• Cell cycle regulation: MEF2A positively affects myoblast proliferation by triggering cell cycle progression through activation of CDK2 protein expression, which may have downstream effects on MYOZ2 function .

This research provides experimental evidence that MEF2A is a positive regulator in skeletal muscle myoblast proliferation and suggests that it regulates myoblast differentiation via MYOZ2 .

What is the relationship between MYOZ2 and muscle fiber type determination?

Studies on the related protein MYOZ1 (calsarcin-2) provide insights into potential MYOZ2 functions:

• Fiber type switching: MYOZ1 knockout mice displayed a switch toward slow-twitch, oxidative fibers, suggesting myozenin family members influence fiber type determination .

• Calcineurin signaling modulation: MYOZ1 KO mice exhibited both increased NFAT activity and enhanced expression of regulator of calcineurin 1-4 (RCAN1-4), indicating enhanced calcineurin signaling in vivo . MYOZ2 likely plays a similar regulatory role in its expression domains.

• Oxidative capacity regulation: Analysis of MYOZ1-deficient muscles showed a 2.6-fold increase in succinate dehydrogenase (SDH)-positive fibers, confirming enhanced oxidative capacity . This suggests myozenin family proteins are involved in metabolic programming of muscle fibers.

• Exercise performance impact: MYOZ1-deficient mice showed markedly improved performance and enhanced running distances in exercise studies . This finding highlights the functional significance of myozenin family proteins in determining muscle performance characteristics.

These discoveries suggest that MYOZ2 likely influences muscle fiber type composition through similar mechanisms in its expression domains, primarily in cardiac muscle and slow-twitch skeletal muscle fibers.