MYO18A Antibody, Biotin conjugated

What is MYO18A and what are its primary functions in cellular biology?

MYO18A (Unconventional myosin-XVIIIa) is a multifunctional protein that serves several critical roles in cellular processes. It may link Golgi membranes to the cytoskeleton and participate in the tensile force required for vesicle budding from the Golgi, thereby playing a role in Golgi membrane trafficking and potentially contributing to the flattened shape of the Golgi apparatus . In concert with LURAP1 and CDC42BPA/CDC42BPB, MYO18A has been implicated in modulating lamellar actomyosin retrograde flow, which is crucial for cell protrusion and migration . Additionally, it functions in maintaining stromal cell architectures required for cell-to-cell contact and regulates trafficking, expression, and activation of innate immune receptors on macrophages . MYO18A also acts as a receptor for surfactant-associated protein A (SFTPA1/SP-A) and plays an important role in the internalization and clearance of SFTPA1-opsonized S. aureus by alveolar macrophages . Notably, it strongly enhances natural killer cell cytotoxicity and suppresses inflammatory responsiveness of macrophages via a mechanism that modulates CD14 trafficking .

What are the key characteristics of the MYO18A Antibody, Biotin conjugated?

MYO18A Antibody, Biotin conjugated is a rabbit polyclonal antibody designed for specific detection of human MYO18A protein . The antibody is typically supplied in a 50μl volume with reactivity specific to human samples . It is derived from rabbit hosts and belongs to the IgG isotype . The primary recommended application for this biotin-conjugated antibody is ELISA, although specific applications may vary between manufacturers . The immunogen used for generating this antibody is typically a recombinant human unconventional myosin-XVIIIa protein fragment corresponding to amino acids 1849-2054 . The biotin conjugation provides enhanced sensitivity and versatility in detection systems, making it compatible with streptavidin-based detection methods .

How does MYO18A function in the immune system?

MYO18A plays several critical roles in immune system function. Research has revealed that it is a novel regulator that limits naïve B cell and immunoglobulin levels while also restricting antigen-induced humoral immunity . Studies using B cell-conditional Myo18A-deficient mice demonstrated that Myo18A deficiency leads to expansion of bone marrow progenitor B cells and mature B cells in secondary lymphoid organs . These mice displayed serum IgM hyperglobulinemia and increased splenic IgM secreting cells, with older mice switching to IgG1 hyperglobulinemia and autoantibody development .

Furthermore, when immunized with inactivated influenza virus, Myo18A-deficient mice developed more potent neutralizing antibodies against the major antigen hemagglutinin, associated with persistent accumulation of antigen-specific germinal center B cells and more antigen-specific bone marrow plasma cells . This indicates that MYO18A functions as a checkpoint regulator in B cell differentiation and antibody production. Additionally, MYO18A regulates trafficking, expression, and activation of innate immune receptors on macrophages and plays a role in suppressing inflammatory responsiveness via CD14 trafficking modulation .

What applications are most suitable for MYO18A Antibody, Biotin conjugated?

The biotin-conjugated MYO18A antibody is primarily recommended for ELISA applications as indicated by product information sheets . The biotin conjugation makes this antibody particularly valuable for detection systems that utilize streptavidin, offering enhanced sensitivity and reduced background compared to non-conjugated primary antibodies. While ELISA is the primary recommended application, researchers may explore its use in other techniques such as immunohistochemistry, immunocytochemistry, or flow cytometry where biotin-streptavidin detection systems are employed, though validation would be necessary before adaptation to these applications. For immunoprecipitation (IP) and Western Blot applications, non-biotin conjugated anti-MYO18A antibodies have been documented to be effective , but the biotin-conjugated version would need validation for these purposes.

What are the recommended storage conditions for maintaining antibody activity?

The MYO18A Antibody, Biotin conjugated should be stored under specific conditions to maintain its activity and specificity. Upon receipt, it is recommended to store the antibody at -20°C or -80°C to maintain long-term stability . Repeated freeze-thaw cycles should be avoided as they can degrade the antibody and reduce its efficacy . The antibody is typically provided in a formulation containing preservatives (0.03% Proclin 300) and stabilizers (50% Glycerol, 0.01M PBS, pH 7.4) . For short-term storage during experimental procedures, the antibody should be kept at 4°C. Proper aliquoting of the antibody upon first thaw is advisable to minimize freeze-thaw cycles and extend the usable life of the product.

What controls should be included when using this antibody in experimental designs?

When designing experiments using MYO18A Antibody, Biotin conjugated, appropriate controls are essential for result validation. The following controls should be considered:

In research settings reported in the literature, controls for MYO18A detection have included rabbit anti-MYO18A antibodies targeting different epitopes and control IgG for immunoprecipitation experiments . The inclusion of these controls ensures experimental rigor and helps distinguish true signals from artifacts.

How can MYO18A Antibody, Biotin conjugated be utilized to study Golgi-cytoskeleton interactions?

Given MYO18A's role in linking Golgi membranes to the cytoskeleton and participating in vesicle budding , the biotin-conjugated antibody can be employed in several advanced applications to investigate these interactions. Researchers can design co-localization studies using the biotin-conjugated MYO18A antibody in combination with antibodies against Golgi markers (e.g., GM130) and cytoskeletal components (e.g., actin, microtubules). The biotin conjugation allows for flexible detection strategies using different streptavidin-conjugated fluorophores to avoid spectral overlap with other fluorescent markers.

For more sophisticated analyses, the antibody could be incorporated into proximity ligation assays (PLA) to detect and quantify interactions between MYO18A and potential binding partners in the Golgi-cytoskeleton interface. Additionally, researchers could combine immunofluorescence with live-cell imaging techniques by first identifying MYO18A-rich regions with the antibody and then tracking vesicle budding events in these regions. When designing such experiments, it is crucial to optimize fixation methods that preserve both Golgi structure and cytoskeletal integrity, as some fixatives may disrupt these interactions.

What methodological approaches can be used to investigate MYO18A's role in B cell differentiation?

Based on the findings that MYO18A functions as a checkpoint regulator in B cell differentiation , several methodological approaches using the biotin-conjugated antibody can be employed:

• Flow Cytometric Analysis: The biotin-conjugated antibody can be used to detect MYO18A expression levels across different B cell developmental stages. This approach would require cell permeabilization protocols to access intracellular MYO18A.

• Immunohistochemistry of Lymphoid Tissues: Biotin-conjugated MYO18A antibody can be used to examine MYO18A distribution patterns in germinal centers, bone marrow, and other lymphoid tissues relevant to B cell development.

• Co-immunoprecipitation Studies: While the biotin-conjugated format is not directly designed for IP, researchers could develop strategies using streptavidin beads to pull down MYO18A and associated proteins to identify interaction partners relevant to B cell signaling pathways.

• Stimulus-Response Experiments: Design protocols to monitor MYO18A localization and expression changes in B cells following stimulation with various activators (e.g., anti-IgM, CD40L, TLR ligands), using the biotin-conjugated antibody for detection.

These methodologies should be implemented with appropriate controls, including comparison with known B cell markers and validation using MYO18A-deficient models when possible.

How does MYO18A deficiency impact antigen-specific antibody responses, and how can this be studied?

Research has demonstrated that MYO18A deficiency leads to more robust antigen-specific B cell antibody responses . When MYO18A-deficient mice were immunized with UV-inactivated influenza virus (UV-PR8), they developed higher serum anti-hemagglutinin (HA) IgM and IgG titers compared to control mice . Furthermore, sera from MYO18A-deficient mice exhibited enhanced neutralizing activity against live PR8 virus in in vitro infection assays .

To further investigate these phenomena, researchers could employ the following methodological approaches:

• Comparative Immunization Studies: Design experiments comparing wild-type and MYO18A-deficient models using various antigens beyond influenza virus to determine if the enhanced antibody response is antigen-specific or a general phenomenon.

• Germinal Center Analysis: Use the biotin-conjugated MYO18A antibody in conjunction with germinal center markers to assess how MYO18A localization and expression correlate with germinal center formation and persistence following immunization.

• Plasma Cell Differentiation Tracking: Develop protocols to monitor the differentiation trajectory from activated B cells to plasma cells in the presence or absence of MYO18A, utilizing the antibody to track expression changes during this process.

• Antibody Affinity Maturation Analysis: Implement methods to assess how MYO18A deficiency affects the affinity maturation process, potentially using surface plasmon resonance (SPR) or similar techniques to measure antibody-antigen binding kinetics.

When designing these studies, it is important to consider the temporal dynamics of immune responses and collect samples at multiple time points post-immunization to capture the full scope of MYO18A's influence on antibody production.

What are the optimal conditions for using MYO18A Antibody, Biotin conjugated in ELISA?

For optimal results when using MYO18A Antibody, Biotin conjugated in ELISA applications, the following methodological considerations are recommended:

While specific dilution recommendations are not provided in the available search results , it is advisable to perform a titration experiment to determine the optimal antibody concentration for your specific application. This should involve testing serial dilutions of the antibody and selecting the concentration that provides the best signal-to-noise ratio.

How can I validate the specificity of MYO18A Antibody, Biotin conjugated in my experimental system?

Validating antibody specificity is crucial for ensuring reliable research results. For MYO18A Antibody, Biotin conjugated, consider implementing the following validation strategies:

• Peptide Competition Assay: Pre-incubate the antibody with excess immunizing peptide (amino acids 1849-2054 of human MYO18A ) before application to your samples. Signal reduction indicates specific binding.

• Knockdown/Knockout Verification: Test the antibody in systems where MYO18A expression has been reduced via siRNA, shRNA, or CRISPR-Cas9. This approach provides definitive evidence of specificity.

• Expression Pattern Analysis: Compare the detection pattern with published MYO18A localization data or with other validated anti-MYO18A antibodies that target different epitopes.

• Cross-Reactivity Assessment: If working with non-human samples, test the antibody against purified MYO18A proteins from different species to confirm cross-reactivity or specificity limitations.

• Western Blot Analysis: Although the biotin-conjugated antibody is primarily recommended for ELISA , Western blot validation could provide additional specificity confirmation by demonstrating recognition of a protein with the expected molecular weight (233 kDa for full-length MYO18A ).

Documentation of these validation steps should be maintained as part of good laboratory practice and can strengthen the credibility of subsequent research findings.

What are the best practices for developing immunofluorescence protocols using this antibody?

While the MYO18A Antibody, Biotin conjugated is primarily recommended for ELISA applications , researchers interested in adapting it for immunofluorescence should consider the following best practices:

• Fixation Optimization: Test multiple fixation methods (4% paraformaldehyde, methanol, or dual fixation) to determine which best preserves MYO18A epitopes while maintaining cellular structure.

• Permeabilization Protocol: Since MYO18A has diverse cellular localizations (Golgi, cytoskeleton, membrane), optimize permeabilization conditions (0.1-0.5% Triton X-100, 0.1-0.5% saponin, or 100% methanol) to ensure antibody access to all relevant compartments.

• Blocking Strategy: Implement robust blocking using 5-10% normal serum from the same species as your secondary reagent, plus 1-3% BSA to minimize background.

• Detection System Selection: Utilize streptavidin conjugated to fluorophores (Alexa Fluor 488, 555, or 647) for detection, selecting wavelengths that complement other fluorophores in your experiment.

• Signal Amplification Consideration: For low-abundance targets, consider tyramide signal amplification (TSA) systems compatible with biotin-streptavidin interactions.

• Dilution Series Testing: Perform a systematic dilution series (typically 1:50-1:500) to identify optimal antibody concentration that balances specific signal with minimal background.

• Confocal Microscopy Settings: When imaging, use appropriate laser power and detector settings to capture MYO18A's diverse localizations without inducing photobleaching or saturating signals.

It is essential to include proper controls, particularly cells known to express MYO18A at varying levels, to establish detection thresholds and confirm specificity in this application.

What are common challenges encountered when using MYO18A Antibody, Biotin conjugated and how can they be addressed?

Researchers working with MYO18A Antibody, Biotin conjugated may encounter several technical challenges. The following table outlines common issues and their solutions:

Implementing a systematic troubleshooting approach that addresses each potential variable sequentially will help identify the source of technical problems and improve experimental outcomes.

How should data from MYO18A studies be analyzed to derive meaningful biological insights?

Analyzing data from studies utilizing MYO18A Antibody, Biotin conjugated requires careful consideration of biological context and appropriate statistical approaches:

When interpreting results, remember that MYO18A functions in multiple cellular processes , so observed effects may reflect its role in various pathways rather than a single mechanism.

How can I integrate MYO18A data with broader immunological datasets to understand its role in immune regulation?

The role of MYO18A as a checkpoint regulator in B cell differentiation positions it within complex immune regulatory networks. To integrate MYO18A data with broader immunological contexts:

• Pathway Analysis: Use bioinformatic tools (KEGG, Reactome, IPA) to place MYO18A findings within known signaling pathways, particularly those involving B cell activation, differentiation, and antibody production.

• Network Visualization: Employ Cytoscape or similar software to visualize interaction networks between MYO18A and other immune regulators, incorporating both your experimental data and published interactions.

• Multi-Omics Integration: Combine MYO18A protein expression data with transcriptomics, epigenomics, or proteomics datasets to identify coordinated regulatory mechanisms. Consider using computational frameworks like MOFA (Multi-Omics Factor Analysis) or DIABLO.

• Phenotype Correlation: Analyze correlations between MYO18A expression/activity and immune phenotypes observed in your models, such as antibody titers, germinal center dynamics, or response to immunization.

• Comparative Analysis: Compare the effects of MYO18A manipulation with those of established immune regulators to position it within known regulatory hierarchies.

• Meta-Analysis Approach: Integrate your findings with published datasets on B cell differentiation factors using meta-analysis techniques to identify consistent patterns across multiple studies.

• Systems Immunology Modeling: Develop mathematical models that incorporate MYO18A within B cell differentiation pathways to predict the systemic effects of its modulation under different immunological challenges.

This integrative approach can reveal how MYO18A functions within the broader immune regulatory landscape and may identify potential points for therapeutic intervention in conditions involving dysregulated antibody responses.

What are the emerging applications for MYO18A Antibody in immunological research?

Based on recent findings regarding MYO18A's role in B cell differentiation and antibody responses , several emerging applications for MYO18A antibodies in immunological research can be anticipated:

• Vaccine Development: Investigating how MYO18A modulation might enhance vaccine-induced antibody responses, given that MYO18A deficiency leads to more potent neutralizing antibodies against viral antigens .

• Autoimmunity Research: Exploring MYO18A's role in preventing autoantibody development, as older MYO18A-deficient mice develop autoantibodies , suggesting potential applications in autoimmune disease research.

• Cancer Immunotherapy: Investigating whether MYO18A manipulation in B cells could enhance anti-tumor antibody responses or influence the tumor microenvironment.

• Germinal Center Biology: Using MYO18A antibodies to study the mechanisms controlling germinal center persistence and selection, which are fundamental to understanding antibody affinity maturation.

• Inflammatory Response Regulation: Examining MYO18A's role in suppressing inflammatory responsiveness of macrophages for potential applications in inflammatory disease research.

These emerging applications highlight the increasing importance of MYO18A antibodies as tools for advancing our understanding of complex immunological processes and potentially developing new therapeutic strategies.

What technical advances might improve the utility of MYO18A antibodies in future research?

Several technical advances could enhance the utility of MYO18A antibodies in research applications:

• Development of Monoclonal Variants: While current MYO18A antibodies are polyclonal , development of monoclonal alternatives targeting specific functional domains would improve reproducibility and enable more precise functional studies.

• Domain-Specific Antibodies: Creating antibodies that specifically recognize different functional domains of MYO18A (motor domain, PDZ domain) would help dissect domain-specific functions.

• Phospho-Specific Antibodies: Developing antibodies that recognize specific phosphorylated states of MYO18A would enable studies of its regulation and activation.

• Improved Conjugation Chemistry: Advanced conjugation techniques that preserve antibody activity while allowing site-specific attachment of biotin or other labels would enhance performance in various applications.

• Humanized Antibodies: For potential therapeutic applications or advanced in vivo imaging, humanized versions of MYO18A antibodies would reduce immunogenicity in human subjects or humanized models.

• Single-Domain Antibodies: Developing smaller antibody formats like nanobodies against MYO18A would improve tissue penetration for in vivo applications and enable super-resolution imaging techniques.