MYH10 Antibody

What is MYH10 and why is it important in cellular research?

MYH10 (myosin heavy chain 10, non-muscle) is a non-muscle myosin II heavy chain protein that plays fundamental roles in cellular contractility, morphology maintenance, adhesion, migration, and division. This 229-231 kDa protein is primarily expressed in nerve cells, megakaryocytes, and various non-muscle cell types . MYH10 forms part of non-muscle myosin II complexes that function as master regulators of actin dynamics—essential for embryogenesis and multiple cellular processes . Recent studies have established MYH10's involvement in pathological conditions, including cancer progression and congenital disorders, making it an important research target .

How do I select the appropriate MYH10 antibody for my research?

When selecting an MYH10 antibody, consider these critical factors:

• Application compatibility: Verify the antibody has been validated for your specific application (WB, IF/ICC, IHC, IP, etc.) and sample type. For example, antibody 21403-1-AP is validated for WB (1:2000-1:10000), IP, IF/ICC (1:10-1:100), and flow cytometry applications .

• Species reactivity: Confirm reactivity with your experimental species. Most commercial MYH10 antibodies react with human, mouse, and rat samples, but cross-reactivity varies by antibody .

• Epitope location: For specific domain studies, select antibodies targeting relevant regions. Some antibodies target C-terminal regions (e.g., ab264266 targets amino acids 1900-C-terminus) , while others target different epitopes.

• Validation evidence: Review published literature citing the antibody and examine validation data provided by manufacturers, including knockout/knockdown controls .

• Antibody format: Most available MYH10 antibodies are rabbit polyclonal, but consider whether your experiment would benefit from monoclonal antibodies for consistent epitope targeting .

What are the optimal sample preparation methods for MYH10 detection in different applications?

Western blotting preparation:

• Use standard cell/tissue lysis buffers containing protease inhibitors to preserve MYH10's high molecular weight (229 kDa)

• Avoid excessive freeze-thaw cycles to prevent protein degradation

• For brain tissue samples (where MYH10 is highly expressed), use 1.0-3.0 mg of total protein lysate with 0.5-4.0 μg of antibody for immunoprecipitation

Immunofluorescence/ICC preparation:

• For cultured cells (e.g., HepG2, HeLa), fix with 4% paraformaldehyde and permeabilize with 0.1% Triton X-100

• When studying MYH10's subcellular localization, particularly in cytoskeletal structures, optimal fixation is critical to preserve structural integrity

• Dilution ratios of 1:10-1:100 are typically recommended for IF/ICC applications

Immunohistochemistry preparation:

• For brain tissue samples, antigen retrieval with TE buffer (pH 9.0) is suggested, though citrate buffer (pH 6.0) may also be used

• For paraffin-embedded tissues, use dilution ratios of 1:50-1:500

How can I optimize MYH10 antibody dilutions for my specific experimental system?

Optimization requires systematic titration based on:

• Start with manufacturer's recommendations: Begin with the suggested dilution range (e.g., 1:2000-1:10000 for WB, 1:10-1:100 for IF/ICC)

• Perform dilution series: Test 3-4 different dilutions within and slightly outside the recommended range

• Assess signal-to-noise ratio: Optimal dilution provides specific signal with minimal background

• Sample-dependent optimization: Adjust based on MYH10 expression level in your specific tissue/cell type. For instance:

  • Brain tissue (high MYH10 expression) may require higher dilutions

  • Cell lines with lower expression may need more concentrated antibody

• Titration methodology: As specified in product documentation, "It is recommended that this reagent should be titrated in each testing system to obtain optimal results."

• Control inclusion: Always include positive controls (tissues/cells known to express MYH10, such as brain tissue or HepG2 cells) and negative controls (secondary antibody alone)

How can I determine the specificity of my MYH10 antibody and avoid cross-reactivity with other myosin isoforms?

Distinguishing MYH10 from other myosin heavy chain proteins (particularly MYH9) requires careful validation:

• Knockout/knockdown verification: Utilize MYH10 knockdown or knockout models as negative controls. Studies have employed CAS9-mediated knockout cells or siRNA-mediated knockdown for specificity validation

• MYH9/MYH10 discrimination: Since MYH9 and MYH10 share high sequence homology, use MYH10-specific antibodies targeting unique regions. For example, antibody 19673-1-AP is specifically designed for MYH10 detection without MYH9 cross-reactivity

• Western blot analysis: Verify a single band at ~229 kDa corresponding to MYH10's molecular weight. Multiple bands may indicate cross-reactivity or degradation

• Double immunostaining: Co-stain with known markers that differentiate MYH10-rich regions from other myosin isoforms. For instance, in tissue sections, MYH10 shows distinct distribution patterns compared to MYH9

• Co-localization studies: In developmental contexts, compare with established expression patterns—MYH10 becomes detectable at the 16-cell stage in mouse embryos while MYH9 is visible from the zygote stage

What are common technical challenges when working with MYH10 antibodies and how can I overcome them?

Challenge 1: High molecular weight detection issues

• Solution: Use low percentage (6-8%) SDS-PAGE gels for adequate separation

• Solution: Extend transfer time for complete transfer of high molecular weight proteins

• Solution: Add SDS (0.1%) to transfer buffer to improve large protein transfer efficiency

Challenge 2: Variable expression levels across tissues

• Solution: Load appropriate protein amounts based on expected expression (e.g., higher amounts for non-neural tissues where MYH10 expression is lower)

• Solution: Adjust exposure times accordingly for different tissue samples

• Solution: Consider MYH10's differential expression pattern—highest in brain tissue with variable expression in other cell types

Challenge 3: Subcellular localization variation

• Solution: For accurate cytoplasmic/membrane localization studies, use proper fixation protocols that preserve cellular architecture

• Solution: Confocal microscopy is recommended for precise localization, as MYH10 co-localizes with other proteins (e.g., MYH9, Snail) in specific cellular compartments

Challenge 4: Maternal vs. zygotic expression confusion in developmental studies

• Solution: Use maternal-zygotic knockout models to distinguish maternal contribution from zygotic expression

• Solution: Time-course analysis can help differentiate expression patterns, as maternal MYH10 products are most abundant in early development

How can MYH10 antibodies be used to study its role in cancer progression and metastasis?

Recent studies highlight MYH10's emerging role in cancer biology, particularly in invasion and metastasis:

What methodological approaches enable the study of MYH10 in developmental and embryonic contexts?

Developmental biology research requires specialized approaches:

• Maternal-zygotic knockout models: Generate maternal-zygotic deletions using Zp3-Cre-mediated maternal deletion of conditional knockout alleles to eliminate maternal contribution of MYH10, crucial for studying early embryonic development

• Time-lapse microscopy: Apply nested time-lapse microscopy to quantitatively assess the effect of maternal-zygotic deletions at different developmental timescales

• Protein-tagging strategies: Use transgenic animals expressing endogenously tagged MYH10 (e.g., MYH10-GFP) to assess relative parental contributions of MYH10 protein during development

• Comparative myosin isoform analysis: Differentiate MYH10 from MYH9 functions using double maternal-zygotic knockouts to reveal compensatory mechanisms between paralogs

• Quantitative developmental phenotyping: Measure specific parameters like contact angles (e.g., 147 ± 2° for wild-type embryos vs. 117 ± 4° for MYH9/MYH10 double knockouts) to assess compaction defects during embryonic development

• In situ hybridization combined with immunostaining: Track tissue-specific expression patterns—at E13.5, MYH10 transcripts are specifically detected in mesenchymal tissue in mouse lungs

How can MYH10 antibodies contribute to understanding extracellular matrix remodeling in disease contexts?

Recent studies reveal MYH10's unexpected role in extracellular matrix (ECM) regulation:

• Thrombospondin expression analysis: MYH10 antibodies can be used to study its relationship with ECM proteins like Thrombospondin. Research shows decreased Thrombospondin expression accompanied by increased matrix metalloproteinase activity in Myh10-deficient lungs

• Matrix metalloproteinase activation studies: Combine MYH10 immunostaining with MMP activity assays to evaluate regulatory relationships. MYH10 deficiency correlates with disrupted ECM remodeling

• ECM protein co-localization: Dual immunofluorescence with MYH10 and ECM protein antibodies can reveal spatial relationships in tissue contexts

• Cell-type specific MYH10 functions: Target mesenchymal-specific expression using conditional knockouts, as loss of MYH10 specifically in mesenchymal cells results in ECM deposition defects and alveolar simplification

• Translational research applications: Compare MYH10 expression in patient samples with ECM disruption phenotypes. MYH10 expression is downregulated in emphysema patients, suggesting potential relevance to human disease

What advanced analytical approaches are recommended for quantifying MYH10 expression across experimental systems?

For rigorous quantitative analysis:

• Multi-platform validation: Cross-validate protein expression using complementary methods (WB, IHC, IF) with appropriate MYH10 antibodies at recommended dilutions (WB: 1:2000-1:10000; IHC: 1:50-1:500; IF: 1:10-1:100)

• Transcript-protein correlation: Combine qPCR for MYH10 mRNA with protein detection to assess post-transcriptional regulation. Primers should be designed for specific amplification of MYH10 transcripts

• Protein stability assessment: Use cycloheximide and proteasome inhibitors (e.g., MG132) with MYH10 antibodies to determine protein half-life and degradation mechanisms, particularly important when studying MYH10's interactions with other proteins

• Subcellular fractionation analysis: Quantify MYH10 distribution across cellular compartments to understand functional relevance in different contexts

• Image-based quantification: For IF/IHC studies, use appropriate software to quantify signal intensity, co-localization coefficients, and spatial distribution patterns

• Reference gene selection: When conducting comparative studies, select appropriate housekeeping controls based on your experimental system and validate their stability across conditions

These methodological approaches provide a robust framework for researchers investigating MYH10 across diverse experimental contexts, from basic mechanistic studies to disease-relevant applications.