MYOZ2 Antibody

What is MYOZ2 and why is it important in muscle research?

MYOZ2 (Myozenin-2, also known as Calsarcin-1 or FATZ-related protein 2) is a protein primarily expressed in cardiac and skeletal muscle tissues where it plays a crucial role in maintaining the structural integrity of muscle fibers . It functions as an intracellular binding protein that links Z-line proteins such as alpha-actinin, gamma-filamin, TCAP/telethonin, and LDB3/ZASP . MYOZ2 also localizes calcineurin signaling to the sarcomere and modulates this signaling pathway . Recent studies have demonstrated its role in myofibrillogenesis and its potential involvement in muscle growth and development . The significance of MYOZ2 extends to pathology, as defects in the MYOZ2 gene are associated with familial hypertrophic cardiomyopathy type 16 (CMH16) .

Which applications are MYOZ2 antibodies most suitable for?

Based on validation data from multiple sources, MYOZ2 antibodies have been successfully employed in the following applications:

The selection of the appropriate application should be guided by your specific research question. For protein expression quantification, WB is most suitable, while IHC and ICC/IF are better for examining localization patterns within tissues or cells .

How should I validate MYOZ2 antibody specificity in my experimental system?

A comprehensive validation strategy for MYOZ2 antibodies should include:

• Positive controls: Use tissues known to express high levels of MYOZ2, particularly heart and skeletal muscle samples .

• Molecular weight verification: Confirm the detected band appears at the expected molecular weight (approximately 30 kDa, though some antibodies may detect bands between 30-35 kDa) .

• Knockdown/overexpression validation: Compare antibody staining in systems with manipulated MYOZ2 expression levels, as demonstrated in studies where MYOZ2 was successfully overexpressed or silenced .

• Cross-reactivity assessment: Test the antibody on tissues from different species to ensure it recognizes the intended target specifically. Most commercial MYOZ2 antibodies have been validated with human, mouse, and/or rat samples .

• Peptide competition assay: Pre-incubate the antibody with the immunizing peptide to confirm binding specificity.

What are the optimal protocols for extracting and preserving MYOZ2 in muscle samples?

For effective MYOZ2 extraction and preservation:

• Tissue collection: Rapidly harvest muscle tissue and either flash-freeze in liquid nitrogen or immediately process for fixation (if intended for histology) .

• Protein extraction for WB:

  • Use RIPA buffer supplemented with protease inhibitors

  • Homogenize tissue thoroughly at 4°C

  • Centrifuge at 12,000g for 15 minutes at 4°C

  • Collect supernatant and determine protein concentration

  • Store aliquots at -80°C to prevent freeze-thaw cycles

• For IHC preparation:

  • Fix tissues in 4% paraformaldehyde

  • For paraffin embedding, suggested antigen retrieval with TE buffer pH 9.0 or alternatively, citrate buffer pH 6.0

  • For frozen sections, fix briefly (10 minutes) in cold acetone

• Cell culture preservation:

  • For myoblasts/myotubes, fix with 4% paraformaldehyde for 15 minutes at room temperature

  • Permeabilize with 0.1% Triton X-100 for 5 minutes

How do I optimize MYOZ2 antibody staining in different muscle types?

Optimization strategies differ between cardiac and skeletal muscle tissues:

For cardiac muscle:

• Use lower antibody dilutions (1:50-1:100) initially for IHC applications

• Extend antigen retrieval time to 20-25 minutes

• Paraffin-embedded human heart tissue has shown good results with MYOZ2 antibodies at 1:300 dilution

For skeletal muscle:

• Higher antibody dilutions (1:100-1:500) are often sufficient

• Include longer blocking steps (1-2 hours) with 5% BSA or 10% normal serum

• For myotubes, DAPI counterstaining helps visualize multinucleated structures

Optimization parameters to test:

• Primary antibody incubation: Test both overnight at 4°C and 1-2 hours at room temperature

• Secondary antibody dilution: Start with 1:500 and adjust as needed

• Washing steps: Increase number and duration for high-background samples

• Signal amplification: Consider using biotin-streptavidin systems for low-abundance detection

How can I investigate the relationship between MEF2A and MYOZ2 in myoblast differentiation?

Research has demonstrated that MEF2A promotes myoblast proliferation and regulates MYOZ2 during myoblast differentiation . To investigate this relationship:

• ChIP assay protocol:

  • Crosslink cells with 1% formaldehyde for 10 minutes at room temperature

  • Lyse cells and sonicate chromatin to 200-500 bp fragments

  • Immunoprecipitate with anti-MEF2A antibody

  • Analyze binding to the MYOZ2 promoter region by qPCR

  • Include IgG controls and positive control regions

• Reporter gene assay:

  • Clone the MYOZ2 proximal promoter into a luciferase reporter vector

  • Co-transfect with MEF2A expression plasmid

  • Measure luciferase activity 24-48 hours post-transfection

  • Include promoter deletion/mutation constructs to identify precise binding sites

• Co-expression analysis:

  • Perform RT-qPCR to monitor MEF2A and MYOZ2 expression during myoblast differentiation

  • Compare with myogenic markers (MyoD, Myf5, myogenin)

  • Western blot to confirm protein level changes

  • Use immunofluorescence to visualize co-localization patterns

• Functional studies:

  • Overexpress or knockdown MEF2A and measure effects on MYOZ2 expression

  • Assess impact on myoblast proliferation using EdU incorporation assays

  • Evaluate differentiation potential through fusion index quantification and myogenic marker expression

What methodologies should I use to study MYOZ2's role in myoblast proliferation versus differentiation?

MYOZ2 exhibits distinct functions during different stages of myogenesis . To investigate these roles:

For proliferation studies:

• MYOZ2 manipulation:

  • Transfect myoblasts with MYOZ2 overexpression plasmid or siRNA

  • Confirm expression changes via RT-qPCR and Western blot (60,000× increase for overexpression, 0.04× for knockdown)

• Proliferation assays:

  • Measure cell viability using CCK8 assay 24-48 hours post-transfection

  • Quantify proliferation via EdU incorporation assay

  • Analyze cell cycle distribution using flow cytometry

• Molecular markers:

  • Assess PAX7 expression (proliferation marker)

  • Monitor MYOD levels (differentiation determining factor)

  • Investigate CDK2 protein expression (cell cycle regulator)

For differentiation studies:

• Differentiation induction:

  • Culture myoblasts to 80-90% confluence

  • Switch to differentiation medium (2% horse serum)

  • Maintain MYOZ2 overexpression or knockdown during differentiation

• Fusion assessment:

  • Quantify myotube formation after 3-5 days

  • Calculate fusion index (nuclei in myotubes/total nuclei)

  • Categorize myotubes by number of nuclei (2-4, 5-10, >10)

• Differentiation markers:

  • Measure early markers (MYF5) and late markers (MYOG)

  • Perform immunofluorescence for muscle-specific proteins like myosin and desmin

How can I design experiments to examine MYOZ2's role in muscle fiber integrity and sarcomere organization?

To investigate MYOZ2's structural functions:

• Ultrastructural analysis:

  • Process muscle samples for transmission electron microscopy (TEM)

  • Fix cells with 2.5% glutaraldehyde in 0.1M sodium cacodylate buffer

  • Post-fix with 1% osmium tetroxide and 1.5% potassium ferrocyanide

  • Measure Z-line width and sarcomere organization

• Co-immunoprecipitation studies:

  • Lyse muscle samples in non-denaturing buffer

  • Immunoprecipitate with anti-MYOZ2 antibody

  • Probe for interaction partners (alpha-actinin, gamma-filamin, TCAP/telethonin, LDB3/ZASP)

  • Confirm reciprocal interactions

• Immunofluorescence co-localization:

  • Double-stain muscle sections or myotubes with MYOZ2 and Z-line markers

  • Acquire high-resolution confocal images

  • Perform quantitative co-localization analysis

  • Use super-resolution microscopy for detailed structural arrangements

• Functional disruption assays:

  • Generate MYOZ2 truncation mutants to identify critical domains

  • Transfect into myotubes and assess effects on sarcomere organization

  • Measure contractile function in engineered muscle tissues

  • Develop MYOZ2 knock-in models with domain-specific mutations

Why might MYOZ2 antibody staining show inconsistent results between experiments?

Several factors can contribute to inconsistent MYOZ2 staining:

• Tissue fixation variations:

  • Overfixation can mask epitopes

  • Underfixation may compromise tissue morphology

  • Standardize fixation time (10-24 hours for tissues, 10-15 minutes for cells)

• Antibody specificity issues:

  • Different antibodies recognize distinct epitopes within MYOZ2

  • Confirm the immunogen region (full-length vs. fragment-specific antibodies)

  • Use antibodies validated for your specific application and species

• Sample-specific factors:

  • MYOZ2 expression varies between muscle types and developmental stages

  • Expression changes during myoblast differentiation (decreases initially then increases)

  • Expression may be altered in disease states or after experimental interventions

• Technical considerations:

  • Antigen retrieval method significantly impacts staining (compare TE buffer pH 9.0 vs. citrate buffer pH 6.0)

  • Secondary antibody cross-reactivity

  • Batch-to-batch variations of antibodies

How should I design experiments to study the epigenetic regulation of MYOZ2?

Based on research showing DNA methylation involvement in MYOZ2 regulation :

• Methylation analysis protocols:

  • Use MethPrimer to predict CpG islands in the MYOZ2 promoter

  • Design bisulfite sequencing PCR (BSP) primers

  • Perform bisulfite conversion of DNA

  • Conduct Sanger sequencing to analyze methylation patterns

• Functional validation approaches:

  • Treat cells with DNA methyltransferase inhibitors (e.g., 5-azacytidine)

  • Measure effects on MYOZ2 expression via RT-qPCR and Western blot

  • Correlate methylation status with expression levels

• Regulatory factor identification:

  • Investigate the role of DNMT1 in MYOZ2 regulation

  • Study lncRNAs that may influence MYOZ2 methylation (e.g., lncMYOZ2)

  • Perform knockdown/overexpression of candidate regulatory factors

• Chromatin organization studies:

  • Conduct ChIP assays to examine histone modifications at the MYOZ2 locus

  • Implement ATAC-seq to assess chromatin accessibility

  • Use 3C/4C/Hi-C techniques to identify long-range chromatin interactions

What are the best approaches for studying MYOZ2 in the context of muscle pathologies?

To investigate MYOZ2 in disease contexts:

• Patient sample analysis:

  • Compare MYOZ2 expression in healthy vs. diseased muscle biopsies

  • Use IHC to examine localization changes in cardiomyopathy samples

  • Perform genetic screening for MYOZ2 mutations in familial cases

• Disease modeling approaches:

  • Generate MYOZ2 mutations associated with cardiomyopathy

  • Develop patient-derived iPSCs for differentiation into cardiomyocytes

  • Analyze structural and functional consequences in 2D and 3D models

• Functional genomics strategies:

  • Perform transcriptomic analysis of MYOZ2-deficient or mutant cells

  • Study the impact of MYOZ2 on calcineurin-NFAT signaling in disease models

  • Examine interaction of MYOZ2 with other Z-line components in pathological states

• Therapeutic investigation platforms:

  • Test compounds that modulate Z-line protein interactions

  • Explore approaches to normalize MYOZ2 expression in deficient systems

  • Develop gene therapy strategies for MYOZ2-related disorders

How can advanced imaging techniques enhance our understanding of MYOZ2 dynamics in muscle cells?

Cutting-edge imaging approaches for MYOZ2 research:

• Live-cell imaging methodologies:

  • Generate MYOZ2-GFP fusion constructs for real-time visualization

  • Utilize FRAP (Fluorescence Recovery After Photobleaching) to measure MYOZ2 mobility

  • Implement TIRF microscopy for Z-line association dynamics

• Super-resolution techniques:

  • Apply STORM or PALM imaging to resolve MYOZ2 nanoscale organization

  • Use SIM for improved visualization of MYOZ2 within the sarcomeric structure

  • Combine with proximity ligation assays to detect protein-protein interactions in situ

• Correlative microscopy approaches:

  • Integrate fluorescence and electron microscopy data

  • Use serial block-face scanning electron microscopy for 3D reconstruction

  • Implement focused ion beam-scanning electron microscopy for ultrastructural context

• Quantitative image analysis:

  • Develop automated algorithms for sarcomere organization assessment

  • Implement deep learning for pattern recognition in MYOZ2 distribution

  • Quantify co-localization coefficients with Z-line markers

What are the most promising techniques for investigating MYOZ2's role in cellular metabolism and signaling?

Recent research has revealed MYOZ2's potential involvement in metabolic regulation :

• Metabolic phenotyping approaches:

  • Measure oxygen consumption rate (OCR) using Seahorse XF Analyzer in MYOZ2-modified cells

  • Assess mitochondrial function parameters (basal respiration, ATP production, maximum capacity)

  • Analyze glycolytic capacity through extracellular acidification rate (ECAR)

• Signaling pathway analysis:

  • Investigate MYOZ2's impact on calcineurin-NFAT signaling

  • Examine phosphorylation status of downstream targets

  • Employ CRISPR-Cas9 screening to identify synthetic interactions

• Protein-protein interaction mapping:

  • Perform BioID or APEX proximity labeling to identify MYOZ2 interactome

  • Validate key interactions through co-immunoprecipitation and FRET

  • Develop domain-specific mutants to disrupt specific interactions

• Translational approaches:

  • Study metabolic changes in muscle-specific MYOZ2 knockout models

  • Investigate MYOZ2's role in insulin response and muscle adaptation

  • Examine potential therapeutic targeting of MYOZ2-mediated pathways