MYBPC3 Antibody

What are the primary applications for MYBPC3 antibodies?

MYBPC3 antibodies are versatile tools that can be utilized in multiple experimental techniques, with varying recommended dilutions for optimal results:

These antibodies have been validated across multiple tissue types, with positive Western blot detection confirmed in mouse, rat, and human heart tissue samples . It is recommended to titrate antibodies in each specific testing system to obtain optimal results, as requirements may be sample-dependent .

What species reactivity can be expected with commercially available MYBPC3 antibodies?

Most commercial MYBPC3 antibodies demonstrate reactivity with samples from multiple species:

• Human samples: Validated in heart tissue and relevant cell lines

• Mouse samples: Particularly heart tissue and specialized cardiomyocyte models

• Rat samples: Primarily heart tissue samples

When selecting a MYBPC3 antibody, review both tested reactivity (experimentally confirmed by the manufacturer) and cited reactivity (reported in published literature) to ensure compatibility with your experimental model . The species reactivity is determined by the conservation of the epitope sequence across species and the specific immunogen used to generate the antibody.

What is the optimal storage protocol for MYBPC3 antibodies?

For maximum shelf life and maintained reactivity:

• Store unopened antibodies at -20°C

• Most formulations remain stable for at least one year after shipment when stored properly

• For many MYBPC3 antibodies, aliquoting is unnecessary for -20°C storage

• Most commercial preparations are supplied in PBS with 0.02% sodium azide and 50% glycerol at pH 7.3

• Some smaller antibody volumes (e.g., 20μl sizes) may contain 0.1% BSA as a stabilizer

Avoid repeated freeze-thaw cycles as they can compromise antibody performance. Always follow manufacturer-specific recommendations, as formulations may vary between suppliers.

How should tissue samples be prepared for optimal MYBPC3 detection?

For immunohistochemistry applications:

• For mouse heart tissue, antigen retrieval with TE buffer at pH 9.0 is typically recommended

• Alternatively, antigen retrieval may be performed with citrate buffer at pH 6.0

• For MYBPC3 detection in fixed tissue samples, a 1:50-1:500 dilution range is typically effective

When working with formalin-fixed, paraffin-embedded samples, thorough deparaffinization and hydration are required before antigen retrieval steps to ensure accessibility of the epitope.

How can MYBPC3 antibodies be used to investigate truncating mutations associated with hypertrophic cardiomyopathy?

MYBPC3 mutations are the most common genetic cause of hypertrophic cardiomyopathy (HCM), with truncating variants accounting for approximately 91% of pathogenic MYBPC3 variants . When designing experiments to investigate these mutations:

• Utilize antibodies targeting different epitopes (N-terminal vs. C-terminal) to distinguish between truncated and full-length proteins

• Implement comparative analysis between patient-derived samples and controls

• Consider combinatorial approaches with genetic testing validation

Research has shown that truncating variants are evenly dispersed throughout the gene, and hypertrophy severity and outcomes are not associated with variant location . This suggests that experimental designs should focus on detecting the presence of truncation rather than its specific location.

What methodological considerations are important when using MYBPC3 antibodies in iPSC-derived cardiomyocyte models?

When employing MYBPC3 antibodies in induced pluripotent stem cell (iPSC) cardiomyocyte models:

• Validation steps should include immunofluorescence confirmation in both heterozygous and homozygous MYBPC3 knockout models

• A homozygous promoter deletion iPSC line can serve as a negative control, as it completely lacks MyBP-C expression by immunofluorescence

• When analyzing frameshift mutations, consider that nonsense-mediated decay (NMD) removes approximately 66-70% of mutant MYBPC3 transcripts in both cellular models and human heart tissue

• For quantitative analysis, normalize protein expression to appropriate loading controls and compare to wild-type reference samples

These considerations ensure reliable interpretation of results when studying MYBPC3 mutations in cellular models.

What are the optimal approaches for using MYBPC3 antibodies in gene therapy research applications?

For researchers investigating AAV-based gene therapies for MYBPC3-associated HCM:

• MYBPC3 antibodies can be used to confirm successful transgene expression following AAV delivery

• Consider using antibodies specific to wild-type MYBPC3 that do not cross-react with common mutant forms

• In AAV9 gene therapy models, antibodies can help verify cardiac-specific expression patterns

• Understand that approximately 72% of MYBPC3-associated HCM patients have AAV9 neutralizing antibody titers ≤1:10, which is relevant for clinical translation

When designing experiments to evaluate gene therapy efficacy, include robust quantification of MYBPC3 protein levels using calibrated Western blot analysis or ELISA techniques with appropriate controls.

How should researchers address epitope-specific considerations when selecting MYBPC3 antibodies?

The structural complexity of MYBPC3 requires careful consideration of epitope location:

• MYBPC3 contains multiple immunoglobulin and fibronectin domains (C0-C10), with nontruncating pathogenic variants clustering particularly in the C3, C6, and C10 domains

• Antibodies targeting the C10 domain may show altered binding patterns in certain mutations, as C10 mutant MyBP-C often fails to incorporate into myofilaments and shows accelerated degradation (≈90% faster)

• In contrast, C3 and C6 mutant MYBPC3 typically incorporates normally with degradation rates similar to wild-type

Therefore, researchers should select antibodies based on the specific domain of interest and the experimental question being addressed. For comprehensive analysis, consider using antibodies targeting different domains simultaneously.

What strategies can resolve weak or absent signal when using MYBPC3 antibodies in Western blot applications?

When facing detection challenges:

• Optimize protein extraction from cardiac tissue:

  • Use specialized extraction buffers containing protease inhibitors

  • Ensure complete homogenization of fibrous cardiac tissue

  • Consider sonication to improve protein solubilization

• Adjust antibody conditions:

  • Increase primary antibody concentration (up to 1:500 dilution)

  • Extend primary antibody incubation time (overnight at 4°C)

  • Test different blocking agents (5% milk vs. 5% BSA)

• Verify protein transfer:

  • MYBPC3 is a large protein (141-150 kDa) that requires extended transfer times

  • Use Ponceau S staining to confirm successful transfer of high molecular weight proteins

  • Consider wet transfer methods for large proteins like MYBPC3

Proper sample preparation is critical, as MYBPC3's association with the cardiac sarcomere can make complete protein extraction challenging.

How can cross-reactivity issues with MYBPC3 antibodies be identified and addressed?

To minimize cross-reactivity concerns:

• Implement proper controls:

  • Include MYBPC3 knockout samples as negative controls

  • Use purified recombinant MYBPC3 protein as a positive control

  • Test antibody specificity in tissues known not to express MYBPC3

• Validation approaches:

  • Perform peptide competition assays to confirm specificity

  • Consider using multiple antibodies targeting different MYBPC3 epitopes

  • Compare reactivity patterns across species with predicted sequence homology

• Experimental design considerations:

  • Increase washing steps duration and frequency

  • Adjust antibody dilution to minimize non-specific binding

  • Pre-absorb antibodies with non-cardiac tissue lysates if non-specific binding persists

These approaches can help distinguish between true MYBPC3 signal and potential cross-reactivity with other MyBP family members or related proteins.

How can MYBPC3 antibodies be utilized in non-invasive biomarker development for hypertrophic cardiomyopathy?

For biomarker development research:

• Sandwich ELISA approaches:

  • Utilize capture and detector antibodies targeting different MYBPC3 epitopes

  • For human MYBPC3 detection, a capture antibody dilution of 2 µg/mL and detector antibody dilution of 0.5 µg/mL has been validated

  • Consider detecting both full-length and truncated MYBPC3 fragments in circulation

• Study design considerations:

  • Include appropriate patient cohorts with confirmed MYBPC3 mutations

  • Correlate circulating MYBPC3 levels with clinical parameters and outcomes

  • Establish normal reference ranges from healthy control populations

• Technical validation:

  • Confirm assay specificity using recombinant MYBPC3 proteins

  • Assess potential interference from other circulating proteins

  • Determine minimum detection limits and dynamic range for clinical utility

This approach leverages MYBPC3 antibodies beyond traditional research applications toward clinical biomarker development for early disease detection and monitoring.

What considerations are important when using MYBPC3 antibodies to investigate posttranslational modifications?

MYBPC3 undergoes significant regulatory phosphorylation, particularly by cAMP-dependent protein kinase (PKA) during adrenergic stimulation . When studying these modifications:

• Selection of phospho-specific antibodies:

  • Choose antibodies that recognize specific phosphorylation sites

  • Validate specificity using phosphatase treatments

  • Consider the impact of sample preparation on phosphorylation status

• Experimental design:

  • Include appropriate controls for basal and stimulated phosphorylation states

  • Carefully time sample collection to capture dynamic phosphorylation events

  • Consider phosphorylation status in the context of disease models

• Technical considerations:

  • Use phosphatase inhibitors during sample preparation

  • Avoid excessive sample heating which may affect phospho-epitopes

  • Consider parallel detection of total and phospho-MYBPC3 for normalization

These approaches enable researchers to investigate how MYBPC3 phosphorylation states relate to cardiac contractility regulation and disease pathogenesis.

How might MYBPC3 antibodies contribute to next-generation gene therapy assessment in cardiomyopathies?

As gene therapy approaches for MYBPC3-associated cardiomyopathies advance:

• Evaluating therapeutic efficacy:

  • Antibody-based quantification of restoration of normal MYBPC3 levels

  • Assessment of proper sarcomeric incorporation of therapeutic MYBPC3

  • Monitoring long-term stability of gene therapy-derived MYBPC3 expression

• Emerging research applications:

  • Development of non-invasive monitoring techniques for gene therapy patients

  • Correlation of MYBPC3 restoration with functional cardiac improvements

  • Identification of potential immune responses to introduced wild-type MYBPC3

• Clinical translation considerations:

  • Understanding that approximately 92% of MYBPC3-associated HCM patients have AAV9 neutralizing antibody titers ≤1:80, indicating potential eligibility for gene therapy

  • Developing companion diagnostics using MYBPC3 antibodies to select appropriate patients

  • Monitoring for immune responses against restored MYBPC3 protein in patients with frameshift mutations

These applications position MYBPC3 antibodies as critical tools in the translational pipeline for novel gene therapies targeting hypertrophic cardiomyopathy.

What role might MYBPC3 antibodies play in understanding the molecular mechanisms of heart failure progression in cardiomyopathy patients?

For investigating disease progression mechanisms:

• Structural and functional changes:

  • Analyze MYBPC3 distribution patterns in different stages of heart failure

  • Assess correlations between MYBPC3 levels/localization and functional parameters

  • Investigate temporal changes in MYBPC3 expression during disease progression

• Interaction studies:

  • Use co-immunoprecipitation with MYBPC3 antibodies to identify altered binding partners

  • Investigate changes in MYBPC3 interactions with actin, myosin, and titin

  • Study how these interactions are modified in different stages of cardiomyopathy

• Mechanistic insights:

  • Determine whether MYBPC3 degradation rates change with disease progression

  • Investigate potential post-translational modifications as disease biomarkers

  • Study compensatory mechanisms in response to MYBPC3 haploinsufficiency

These applications can provide deeper understanding of how MYBPC3 mutations lead to progressive cardiac dysfunction, potentially identifying new therapeutic targets beyond gene replacement.