MYO1A Antibody

What is MYO1A and why is it an important research target?

MYO1A (Myosin IA) is an unconventional myosin that functions as an actin-based molecular motor. It plays critical roles in directing the movement of organelles along actin filaments . Within intestinal epithelial cells, MYO1A is abundantly expressed in the brush border microvilli where it functions at the interface between membrane and actin cytoskeleton . By coupling actin dynamics with cellular signaling, MYO1A facilitates the organization and renewal of apical structures in epithelial cells, supporting vital transport and barrier functions . Research has associated MYO1A mutations with autosomal dominant deafness (DFNA48) , making it an important target for both basic science and clinical research applications.

Verifying antibody specificity is crucial for reliable research outcomes. Follow these methodological approaches:

• Knockout validation: Use tissue or cells from MYO1A knockout models as negative controls. Research has shown that MYO1A KO mice demonstrate no MYO1A signal in the brush border region when immunostained with anti-MYO1A antibodies .

• Molecular weight verification: Confirm that your antibody detects the expected molecular weight band (~118-130 kDa) on Western blots .

• Cross-reactivity testing: Verify reactivity against MYO1A from different species based on your experimental design, as some antibodies have been validated for human, mouse, and rat samples .

• Orthogonal validation: Compare results with different antibodies targeting distinct epitopes of MYO1A, or use alternative detection methods like RNAseq .

• Positive control testing: Use tissues or cell lines known to express MYO1A, such as intestinal epithelial cells, HepG2, HeLa, or A431 cells .

What are the optimal conditions for using MYO1A antibodies in different applications?

Based on extensive validation studies, the following application-specific conditions are recommended:

Western Blot (WB):

• Dilution range: 1:500-1:2000

• Recommended positive controls: HepG2, HeLa, A431 cells, mouse brain tissue

• Expected molecular weight: 118-130 kDa

Immunohistochemistry (IHC):

• Dilution range: 1:50-1:500

• Recommended antigen retrieval: TE buffer pH 9.0 or citrate buffer pH 6.0

• Positive control tissues: Human colon carcinoma, human kidney tissue

Immunofluorescence (IF)/Immunocytochemistry (ICC):

• Dilution range: 1:50-1:500

• Recommended cell types: HepG2, HeLa cells

Immunoprecipitation (IP):

• Antibody amount: 0.5-4.0 μg for 1.0-3.0 mg of total protein lysate

• Validated in: HeLa cells

Flow Cytometry (FCM):

• Dilution range: 1:10-1:50

How should I optimize MYO1A antibody staining in microvilli-rich tissues?

Optimizing MYO1A antibody staining in microvilli-rich tissues requires special consideration due to the complex structure of these cellular domains:

• Fixation method: For brush border microvilli visualization, use 4% paraformaldehyde fixation to preserve microvillar structure .

• Co-staining strategy: Combine MYO1A antibody with F-actin markers (phalloidin) to clearly define microvillar structures . This approach enhances the accuracy of MYO1A localization analysis.

• Quantification method: For quantitative analysis of MYO1A enrichment in microvilli, use the Fold Enrichment in Microvilli (FEM) method:

  • Identify microvilli using F-actin (phalloidin) channel

  • Measure MYO1A intensity at 5-7 points mapping to microvillar F-actin labeling

  • Measure intensity at 5-7 points in non-nuclear cytosolic regions

  • Calculate microvilli/cytosol intensity ratio (FEM)

• Competing epitopes consideration: Be aware that certain phospholipid-binding proteins (particularly lactadherin-C2, a phosphatidylserine-binding protein) may compete with MYO1A for binding sites in microvilli, potentially affecting staining patterns .

What are the critical storage and handling conditions for maintaining MYO1A antibody efficacy?

Proper storage and handling are essential for maintaining antibody performance:

• Storage temperature: Store antibodies at -20°C for long-term preservation .

• Aliquoting strategy: Divide antibodies into small aliquots to avoid repeated freeze/thaw cycles that can compromise antibody function .

• Buffer composition: Most MYO1A antibodies are supplied in PBS containing protectants such as:

  • 0.02-0.09% sodium azide

  • 50% glycerol (pH 7.3)

  • Sometimes includes BSA (0.1%) in smaller volume preparations

• Stability timeline: When stored properly at -20°C, MYO1A antibodies typically remain stable for one year after shipment .

• Working solution handling: For diluted working solutions, prepare fresh and maintain at 4°C for short-term use only.

How can I address non-specific binding or high background issues with MYO1A antibodies?

Non-specific binding and high background are common challenges when working with MYO1A antibodies. Address these methodologically:

• Blocking optimization: For membrane-associated proteins like MYO1A, use specialized blocking reagents such as those recommended for phospholipid-binding proteins. Consider 5% BSA in TBS-T rather than milk-based blockers, as milk contains bioactive lipids that may interfere with lipid-binding domains of MYO1A .

• Dilution titration: Systematically test antibody dilutions across a wider range than recommended (e.g., extend to 1:5000 for WB) to identify the optimal signal-to-noise ratio for your specific experimental system .

• Secondary antibody cross-reactivity: Verify that your secondary antibody does not cross-react with endogenous immunoglobulins in your sample. For intestinal tissue sections, this is particularly important due to the presence of immune cells .

• Antigen retrieval method comparison: For fixed tissues, compare different antigen retrieval methods (citrate buffer pH 6.0 vs. TE buffer pH 9.0) as MYO1A epitope accessibility may vary depending on the retrieval approach .

• Negative control implementation: Include knockout tissues/cells or primary antibody omission controls to distinguish between specific and non-specific signals.

How should I interpret differences between expected and observed molecular weights for MYO1A in Western blots?

MYO1A has a calculated molecular weight of 118 kDa, but is often observed at 130 kDa on Western blots . These discrepancies require careful interpretation:

• Post-translational modifications: MYO1A is subject to phosphorylation by ALPK1 , which can increase its apparent molecular weight. Consider using phosphatase treatment of lysates to determine if this explains the size shift.

• Isoform variation: Alternative splicing of the MYO1A gene has been reported , potentially resulting in different protein variants with altered molecular weights.

• Species-specific differences: Compare observed molecular weights across species carefully, as there may be species-specific post-translational modifications or splice variants.

• SDS-PAGE conditions: The migration pattern of MYO1A can be affected by gel percentage, buffer systems, and running conditions. Use 5% SDS-PAGE for optimal separation of high molecular weight proteins like MYO1A .

• Sample preparation influence: The method of sample preparation (denaturing conditions, reducing agents) can affect the conformation of MYO1A and its migration pattern in gels.

How can I differentiate between MYO1A and other myosin family members in my experiments?

Myosin family members share structural similarities, making specific detection challenging. Use these methodological approaches:

• Epitope selection strategy: Choose antibodies targeting the TH1 domain (tail homology 1), which is more divergent between myosin classes than the head domain .

• Knockout validation: In MYO1A knockout models, verify that your antibody shows no signal, confirming specificity .

• Redundancy analysis: Be aware that other myosin family members may be upregulated in MYO1A knockout models. For example, myosin-1c (Myo1c) shows significant recruitment into the brush border of MYO1A KO enterocytes, indicating functional redundancy .

• Subcellular localization comparison: Different myosin family members show distinct subcellular localization patterns. MYO1A is enriched in microvilli, while other myosins may show different distribution patterns .

• Immunoprecipitation followed by mass spectrometry: For definitive identification, consider immunoprecipitating with your antibody followed by mass spectrometry analysis to confirm the captured protein's identity.

How can I use MYO1A antibodies to study membrane-cytoskeleton interactions in epithelial cells?

MYO1A serves as an important linker between the plasma membrane and actin cytoskeleton in epithelial cells, particularly in microvilli. To study these interactions:

• Co-localization analysis protocol: Use dual immunofluorescence with MYO1A antibodies (1:50-1:500 dilution) and membrane markers or actin cytoskeleton probes (phalloidin) . This approach allows visualization of MYO1A at the membrane-cytoskeleton interface.

• Membrane binding domain investigation: MYO1A contains multiple membrane binding motifs within its TH1 domain, including the N-terminal targeting motif (NTM) and C-terminal targeting motif (CTM) . Use antibodies recognizing these specific domains to study their contributions to membrane localization.

• Lipid interaction analysis: Combine MYO1A immunostaining with specific lipid-binding probes like lactadherin-C2 (phosphatidylserine) or PLCδ1-PH (phosphoinositol 4,5-bisphosphate) to study MYO1A association with specific membrane phospholipids .

• Live cell imaging approach: For dynamic studies, use antibodies against MYO1A fused with fluorescent proteins (e.g., 3x-mCitrine-Myo1a-TH1) and employ single molecule TIRF microscopy to observe membrane detachment rates and dynamics .

• Membrane tension studies: MYO1A regulates microvillar membrane tension . Use antibodies to study MYO1A distribution under conditions that alter membrane tension, such as osmotic challenges or cytoskeletal perturbations.

What approaches can I use to study MYO1A in disease models, particularly intestinal disorders?

MYO1A's critical role in brush border structure and function makes it relevant for intestinal disease research:

• Comparative expression analysis: Use immunohistochemistry (1:50-1:500 dilution) to compare MYO1A expression patterns between normal intestinal tissue and disease models . Focus on colon carcinoma tissues, which have been validated for MYO1A antibody staining .

• Brush border structural analysis: In disease models, assess brush border structural integrity using co-staining of MYO1A with other brush border components such as sucrase-isomaltase (SI) or alkaline phosphatase (AP) .

• Functional recovery assessment: In recovery or treatment models, monitor MYO1A re-localization to the brush border as a marker for epithelial differentiation and functional restoration.

• Vesicle release dynamics: MYO1A plays a role in vesicle release from microvillar tips, which may impact gut host defense . Use antibodies to track changes in this process under disease conditions.

• Genetic variant analysis: For hereditary intestinal disorders, use antibodies that can detect common MYO1A variants or mutations to assess their expression and localization patterns.

How can I effectively use MYO1A antibodies in studies of hearing loss mechanisms?

MYO1A mutations have been associated with autosomal dominant deafness (DFNA48) , making it relevant for hearing research:

• Inner ear tissue preparation protocol: For cochlear sections, use specialized fixation (4% PFA, 2 hours) followed by decalcification before proceeding with standard immunohistochemistry protocols using MYO1A antibodies (1:50-1:500) .

• Hair cell stereocilia analysis: Focus on stereocilia of cochlear hair cells, where myosins play important structural and functional roles. Use MYO1A antibodies in combination with F-actin markers to assess potential stereociliary localization and ultrastructure.

• Genetic mutation model investigation: In models harboring specific MYO1A mutations associated with DFNA48, use antibodies that can still recognize the mutated protein to assess changes in expression, localization, or protein stability.

• Cross-reactivity considerations: Be aware that multiple myosins are expressed in hair cells. Verify antibody specificity in cochlear tissues using appropriate controls to ensure you're specifically detecting MYO1A and not related myosins.

• Functional correlation approach: Correlate MYO1A immunolocalization with auditory testing results to establish relationships between protein distribution patterns and functional deficits in hearing models.

How might emerging techniques enhance MYO1A antibody applications in research?

Emerging technologies offer new possibilities for MYO1A antibody applications:

• Super-resolution microscopy approaches: Techniques like STORM or PALM can resolve MYO1A distribution within microvillar subdomains at nanometer resolution, providing insights into molecular organization not visible with conventional microscopy .

• Proximity labeling methods: Using antibodies in conjunction with enzymatic proximity labeling (BioID, APEX) can identify novel MYO1A-interacting proteins within specific cellular compartments.

• Single-cell proteomics integration: Combining immunofluorescence data with single-cell proteomics allows correlation of MYO1A expression levels with global proteome changes across individual cells in heterogeneous populations.

• Cryo-electron tomography application: Using antibodies conjugated to gold particles for cryo-ET can reveal the 3D architecture of MYO1A in relation to membrane and cytoskeletal components at molecular resolution.

• Organoid-based screening platforms: Testing MYO1A antibodies in intestinal organoids provides a physiologically relevant system for studying protein dynamics in a 3D context that better mimics in vivo conditions.

What are the current challenges in developing more specific MYO1A antibodies for research applications?

Despite advances in antibody technology, several challenges remain in developing highly specific MYO1A antibodies:

• Epitope accessibility limitations: MYO1A's membrane association and complex conformational states can limit epitope accessibility. Future antibody development should target epitopes that remain accessible in native conditions .

• Cross-reactivity with related myosins: The structural similarity between MYO1A and other class I myosins complicates the development of truly specific antibodies. More sophisticated immunogen design focusing on highly divergent regions is needed.

• Species conservation challenges: High conservation of MYO1A across species makes it difficult to generate antibodies that recognize species-specific variants, which is important for comparative studies.

• Post-translational modification detection: Current antibodies rarely distinguish between modified (e.g., phosphorylated) and unmodified forms of MYO1A . Developing modification-specific antibodies would enhance functional studies.

• Conformational state recognition: Generating antibodies that specifically recognize different conformational states (e.g., membrane-bound vs. cytosolic) would enable more nuanced studies of MYO1A dynamics and function.

How might MYO1A antibodies contribute to translational research in epithelial biology and disease?

MYO1A antibodies have significant potential in translational research:

• Diagnostic biomarker development: Changes in MYO1A expression or localization could serve as biomarkers for epithelial differentiation states or intestinal pathologies. Standardized immunohistochemistry protocols using validated antibodies would be essential for clinical application .

• Therapeutic response monitoring: MYO1A antibodies could be used to assess restoration of normal epithelial organization following therapeutic interventions in intestinal disorders.

• Drug screening platform enhancement: High-content screening using MYO1A antibodies could identify compounds that restore normal microvillar organization in disease models.

• Personalized medicine approaches: In patients with MYO1A mutations, antibodies that can distinguish between normal and mutant proteins could help predict disease progression or treatment response.

• Regenerative medicine applications: During intestinal regeneration or stem cell differentiation, MYO1A antibodies could serve as markers for monitoring proper epithelial polarization and brush border formation, critical for functional restoration.