Myh9 Antibody

What is MYH9 and why is it important in research?

MYH9 (myosin heavy chain 9) is a non-muscle myosin protein that functions as a critical component of the actin cytoskeleton. The protein is encoded by the MYH9 gene located on chromosome 22q12.3, spanning over 106 kilobases with 41 exons that translate into a 1,960 amino acid protein . With a molecular weight of approximately 226.5 kDa, MYH9 forms part of a hexameric complex consisting of two heavy chains, two regulatory light chains, and two essential light chains .

MYH9 plays essential roles in cellular processes including:

• Cell migration and adhesion

• Cytokinesis and cell division

• Maintenance of cell shape

• Specialized functions like secretion and capping

• Cytoskeletal reorganization

Research interest in MYH9 has intensified due to its involvement in multiple pathological conditions, particularly cancer progression and rare genetic disorders.

What applications are MYH9 antibodies most commonly used for?

MYH9 antibodies are versatile tools employed across multiple research applications with varying recommended dilutions:

Successful applications vary by antibody source and experimental design. Many commercially available antibodies have been validated in cell lines such as A549, HeLa, SGC-7901, MDCK, and 293T cells .

How should researchers select appropriate positive controls for MYH9 antibody validation?

Proper validation requires selection of appropriate positive controls. Based on validated literature and manufacturer recommendations, these cell lines and tissues consistently show MYH9 expression:

Recommended cell lines:

• 293T, A431, HeLa, HepG2 (human)

• NIH-3T3, BCL-1, Raw264.7, C2C12 (mouse)

• Rat2 (rat)

Recommended tissues:

• Human and mouse kidney tissue (particularly with antigen retrieval using TE buffer pH 9.0)

Always perform both positive and negative controls, including using lysates from MYH9 knockout or knockdown cells where feasible, to ensure antibody specificity.

What factors affect MYH9 antibody specificity and how can researchers ensure optimal results?

Several factors impact MYH9 antibody performance, requiring careful consideration:

Epitope recognition region:
Different antibodies target distinct regions of MYH9. For example, some target the N-terminal region , middle region , or C-terminal region , potentially affecting cross-reactivity and application performance.

Host species and antibody type:
MYH9 antibodies are available as:

• Rabbit monoclonal (highest specificity, often recombinant)

• Rabbit polyclonal (greater epitope coverage)

• Mouse monoclonal (clone-specific recognition)

Cross-reactivity considerations:
Some MYH9 antibodies may cross-react with other myosin family members. For instance, the PA5-17025 antibody specifically does not cross-react with nonmuscle heavy chains of myosin IIb or IIc , making it valuable for discriminating between myosin isoforms.

Methodological recommendations:

• Validate antibodies using multiple techniques (WB, IF, IHC)

• Include both positive and negative controls

• Consider using siRNA knockdown validation (can reduce MYH9 expression by ~70%)

• For protein interaction studies, compare results from native and denatured immunoprecipitation conditions

What are the optimal immunohistochemistry protocols for MYH9 detection?

Successful MYH9 immunohistochemical detection requires careful attention to protocol details:

Tissue preparation and fixation:

• Formalin-fixed, paraffin-embedded (FFPE) tissues are commonly used

• Fresh-frozen sections may provide better epitope preservation

Antigen retrieval considerations:

• Primary recommendation: TE buffer pH 9.0

• Alternative: Citrate buffer pH 6.0

Primary antibody incubation:

• Typical dilutions range from 1:50-1:500 depending on antibody source

• Recommended incubation: Overnight at 4°C or 1-2 hours at room temperature

Detection systems:

• HRP-polymer detection systems typically yield cleaner results with less background

• For fluorescent detection, select secondary antibodies with minimal cross-reactivity

Counterstains:

• Hematoxylin (for brightfield)

• DAPI (for fluorescence)

Researchers should note that MYH9 expression exceeds 80% in human lung cancer tissues and adjacent tissues , making it an important diagnostic consideration in cancer research applications.

How do storage conditions affect MYH9 antibody performance and stability?

Maintaining antibody integrity requires proper storage and handling:

Recommended storage conditions:

• Temperature: Most MYH9 antibodies should be stored at -20°C

• Buffer composition: Typically PBS with 0.02% sodium azide and 50% glycerol pH 7.3

• Avoid repeated freeze-thaw cycles (aliquoting may be necessary for some products)

Special handling notes:

• Some antibodies (e.g., ZRB1165) specify storage as a concentrated solution with brief centrifugation prior to opening

• For PA5-17025, aliquoting is specifically not recommended

Stability considerations:

• Most antibodies maintain stability for one year after shipment when properly stored

• Always check manufacturer guidelines for specific products

• Document lot numbers and validation dates for experimental reproducibility

How can MYH9 antibodies be utilized to study cancer progression mechanisms?

Recent research has revealed MYH9's critical role in cancer progression across multiple tumor types:

MYH9 in glioma progression:
Studies have established that MYH9 promotes epithelial-mesenchymal transition (EMT) signaling in glioma cells. Knockdown experiments demonstrate that silencing MYH9 significantly inhibits tumor cell migration and invasion capacity, with concurrent reductions in β-catenin and N-cadherin expression and increases in E-cadherin .

Molecular interaction mechanisms:
MYH9 forms a complex with β-catenin and USP2 deubiquitinase, which:

• Stabilizes β-catenin by preventing ubiquitin-mediated degradation

• Promotes EMT signaling cascade activation

• Enhances tumor cell invasion and metastasis

Experimental approaches:

• Coimmunoprecipitation (Co-IP) to detect MYH9 interactions with β-catenin and USP2

• Immunofluorescence colocalization studies (showing primarily cytoplasmic localization)

• Cycloheximide (CHX) chase assays to evaluate protein stability

• Transwell migration and Boyden invasion assays to assess functional consequences

Researchers investigating these pathways should consider combined approaches using MYH9 antibodies alongside genetic knockdown methodologies for comprehensive mechanistic insights.

What methodologies are effective for studying MYH9 knockdown effects alongside antibody-based detection?

Combining genetic manipulation with antibody detection provides powerful mechanistic insights:

siRNA and shRNA approaches:

• Short hairpin RNA (shRNA) lentiviral constructs targeting MYH9 (demonstrated in U87 and U251 glioma cells)

• Small interfering RNAs (siRNAs) targeting MYH9 (70% knockdown efficiency reported)

Experimental workflow:

• Transfect cells with MYH9 siRNA/shRNA or negative controls (siNC/shNC)

• Validate knockdown efficiency by RT-qPCR and Western blot (36-60h post-transfection)

• Perform functional assays (migration, invasion, proliferation)

• Use MYH9 antibodies to assess downstream signaling effects

Quantifiable parameters:
Research shows MYH9 knockdown can:

• Reduce cell migration by ~25%

• Inhibit cancer cell growth by up to 70%

• Alter EMT marker expression (decreased β-catenin and N-cadherin, increased E-cadherin)

Technical considerations:

• Transfection efficiency varies by cell line (typically use Lipofectamine 3000)

• Medium should be changed 8 hours post-transfection

• Collect cells at 36 hours (RNA analysis) and 60 hours (protein analysis) post-transfection

How do MYH9 antibodies contribute to understanding therapeutic approaches in cancer research?

MYH9 antibodies are valuable tools for investigating potential therapeutic interventions:

Anti-tumor effects:
Recent research demonstrates that MYH9 antibodies can directly inhibit cancer cell growth. At a concentration of 300 μM, antibody MYH9 inhibited cell growth by 30% and reduced migration rate by 25% . This is particularly relevant for developing targeted therapies.

Combinatorial approaches:
Combining MYH9-targeted treatments with conventional therapeutics shows promise:

• Lidamycin (LDM) antibiotic combined with MYH9 antibody demonstrated enhanced efficacy

• In H460 cells, 2 nM LDM induced apoptosis in 70% of cells

• LDM downregulated B-cell lymphoma-2 (Bcl-2) and nuclear factor kappa-B (NF-κB) levels

In vivo evidence:
LDM at different concentrations showed significant inhibitory effects on:

• Human large cell lung cancer H460 xenograft tumors (53.20% and 69.80%)

• Lung adenocarcinoma xenograft tumors (40.20% and 58.30%)

These findings suggest MYH9 antibodies not only serve as research tools but potentially as therapeutic agents or for identifying patients who might benefit from targeted therapies.

How can researchers effectively study MYH9-related diseases using antibody-based approaches?

MYH9 mutations lead to a spectrum of autosomal dominant disorders collectively termed MYH9-related diseases (MYH9-RD), including May-Hegglin anomaly, Fechtner syndrome, and Epstein syndrome . These conditions share features including macrothrombocytopenia, with variable manifestations of hearing loss, renal failure, and cataracts.

Research design considerations:

• Patient sample collection (blood, platelets, kidney biopsies)

• Genetic screening for MYH9 mutations

• Antibody-based detection of protein expression/localization abnormalities

Methodological approaches:

• Platelet analysis:

  • Immunofluorescence staining of peripheral blood smears

  • Assessment of MYH9 protein inclusion bodies in neutrophils

• Kidney pathology:

  • Immunohistochemistry of renal biopsies using anti-MYH9 antibodies

  • Correlation of MYH9 distribution with glomerular damage

• Cell models:

  • Creation of patient-derived cell lines

  • CRISPR-based introduction of MYH9 mutations

Technical recommendations:

• Use of multiple antibodies targeting different MYH9 regions

• Combination with genetic testing and clinical correlation

• Detailed morphological analysis alongside immunostaining

What are the best practices for studying MYH9 protein-protein interactions?

Investigating MYH9's interactions with other proteins requires sophisticated approaches:

Recommended co-immunoprecipitation (Co-IP) methods:

• Native conditions:

  • Preserve protein complexes in their natural state

  • Useful for identifying physiological interaction partners

  • Buffer considerations: RIPA lysis buffer containing PMSF and phosphatase inhibitors (100:1:1 ratio)

• Denatured conditions:

  • Reduces non-specific interactions

  • Helps confirm direct protein binding

  • Particularly useful when validating novel interactions

Structural domain analysis:
Research has identified three main MYH9 domains:

• Muscle ball tail domain

• Muscle ball head movement domain

• Muscle N domain

Interaction mapping studies reveal that the myosin tail domain binds to β-catenin , providing insights into functional consequences of these interactions.

Advanced techniques:

• Proximity ligation assays for detecting protein interactions in situ

• FRET-based approaches for real-time interaction monitoring

• Mass spectrometry following immunoprecipitation for unbiased interaction screening

How do different epitope targets affect MYH9 antibody performance in research applications?

MYH9 antibodies targeting different protein regions demonstrate varying performance characteristics:

Epitope mapping considerations:

• N-terminal antibodies (e.g., GeneTex anti-MYH9 antibody [N1-2])

• Middle region antibodies (e.g., Aviva Systems Biology MYH9 antibody)

• C-terminal antibodies (e.g., Sigma anti-Myosin-9/MYH9 antibody targeting epitope within 16 amino acids from C-terminus)

Performance comparison:

Application-specific considerations:

• For protein interaction studies, select antibodies targeting regions unlikely to be involved in binding interfaces

• For detecting specific mutations, choose antibodies recognizing regions distant from the mutation site

• For phosphorylation studies, consider antibodies that aren't affected by phosphorylation state

The immunogen sequence can significantly impact antibody performance. For example, one antibody uses an immunogen with sequence: REQEVNILKKTLEEEAKTHEAQIQEMRQKHSQAVEELAEQLEQTKRVKANLEKAKQTLENERGELANEVKVLLQGKGDSEHKRKKVEAQLQELQVKFNEGERVRTELADKVTKLQVELDNVTGLLSQSDSKSSKLTKDF .

What approaches can resolve common technical challenges when working with MYH9 antibodies?

Researchers frequently encounter several challenges when working with MYH9 antibodies:

Challenge: High molecular weight detection issues
Solution:

• Use gradient gels (4-15%) for better separation of high molecular weight proteins

• Extend transfer time (overnight at lower voltage) for complete transfer

• Use PVDF membranes instead of nitrocellulose for improved binding of high MW proteins

Challenge: Background in immunostaining
Solutions:

• Optimize blocking (5% BSA often more effective than milk for MYH9)

• Increase wash steps (at least 3×10 minutes between antibody incubations)

• Titrate primary antibody concentration (start with manufacturer recommendations)

• For tissues, consider antigen retrieval optimization (compare citrate pH 6.0 vs. TE pH 9.0)

Challenge: Inconsistent knockdown validation
Solutions:

• Ensure primers/antibodies don't overlap with siRNA targeting region

• Validate at both RNA (36h) and protein (60h) levels

• Include multiple siRNA constructs to confirm specificity

Challenge: Co-IP difficulties
Solutions:

• Try both native and denatured conditions

• Use DSP crosslinking to stabilize transient interactions

• Consider the buffer composition (RIPA with PMSF and phosphatase inhibitors at 100:1:1 ratio)

How can researchers optimize experiments comparing MYH9 expression across different sample types?

Comparative studies require careful standardization:

Sample preparation standardization:

• Use consistent cell densities and lysis protocols

• For tissues, employ laser capture microdissection to isolate specific cell populations

• Match protein concentrations precisely before loading

Quantification approaches:

• Utilize loading controls appropriate for your experimental context:

  • GAPDH (general cytoplasmic)

  • β-actin (may be problematic due to interaction with myosins)

  • Total protein stains (REVERT or similar) for whole tissue lysates

Technical considerations:

• Run samples to be compared on the same gel/blot

• Use biological and technical replicates (minimum n=3)

• Apply quantitative analysis (densitometry with normalization)

• Consider qPCR validation of protein expression findings