MYO3B Antibody, HRP conjugated

What is MYO3B and what cellular functions does it perform?

MYO3B (Myosin-IIIb) is a 151.8 kDa protein belonging to the STE Ser/Thr protein kinase family with a canonical length of 1341 amino acid residues in humans. It functions as an actin-based motor protein with protein kinase activity and is primarily localized in the cytoplasm. The protein is expressed predominantly in the retina, kidney, and testis tissues. MYO3B serves as a cellular marker for identifying Midbrain-Derived Inhibitory Neurons in certain research contexts .

The protein is characterized by its dual functionality - combining both motor protein capabilities and enzymatic kinase activity. This unique combination allows MYO3B to participate in cellular processes requiring both mechanical force generation and signaling pathway regulation. Understanding this dual functionality is crucial for correctly interpreting experimental results when using MYO3B antibodies in research settings .

How does HRP conjugation affect antibody performance in various applications?

HRP (horseradish peroxidase) conjugation provides a reliable enzymatic detection mechanism that enables quantitative and qualitative analysis in various immunoassays. For MYO3B antibodies specifically, HRP conjugation maintains the antibody's specificity while providing enhanced detection sensitivity in ELISA applications . The conjugation process preserves the antibody's binding characteristics to the target protein while adding the enzymatic capability necessary for colorimetric or chemiluminescent detection systems .

What are the specific experimental applications validated for MYO3B Antibody, HRP conjugated?

Based on manufacturer specifications, MYO3B Antibody, HRP conjugated has been primarily validated for ELISA applications . The antibody demonstrates reactivity specifically with human samples, as it was developed using recombinant human Myosin-IIIb protein (amino acids 1110-1276) as the immunogen . While ELISA represents the validated application, researchers should note that optimization may be required for specific experimental conditions.

How should researchers optimize experimental protocols for MYO3B detection in different tissue types?

When optimizing protocols for MYO3B detection across different tissue types, researchers should consider tissue-specific expression patterns. MYO3B is predominantly expressed in retina, kidney, and testis, which may require different sample preparation approaches . For retinal tissues, specialized fixation protocols that preserve both protein structure and tissue architecture are recommended, while kidney and testis samples may benefit from different detergent compositions during extraction.

Tissue-specific optimization should include:

• Sample preparation modifications: Adjust buffer compositions based on tissue lipid content and cellular density

• Incubation time variations: Extend primary antibody incubation for tissues with lower MYO3B expression

• Blocking reagent selection: Use tissue-matched blocking solutions to minimize background

• Signal amplification: Consider supplementary detection systems for tissues with minimal expression

For quantitative analyses, researchers should develop tissue-specific standard curves using recombinant MYO3B protein to account for matrix effects from different tissue environments . Additionally, cross-validation with unconjugated antibodies may help distinguish between detection limitations and actual biological variations in MYO3B expression patterns .

What methodological approaches can address cross-reactivity concerns when studying MYO3B in systems with multiple myosin isoforms?

To minimize cross-reactivity issues, researchers should implement:

• Pre-absorption controls: Incubate antibody with recombinant proteins of potentially cross-reactive myosin isoforms prior to immunodetection

• Peptide competition assays: Conduct parallel experiments with and without specific blocking peptides

• Knockout/knockdown validation: When possible, utilize MYO3B-depleted samples as negative controls

• Orthogonal detection methods: Confirm findings using alternative detection approaches with different epitope targets

Particular attention should be paid to distinguishing MYO3B from MYH3 and other myosin family members that share structural similarities . Researchers should compare sequence homology between the immunogen sequence and other myosin isoforms to predict potential cross-reactivity and design appropriate control experiments accordingly .

How does the polyclonal nature of available MYO3B antibodies affect experimental reproducibility?

The polyclonal nature of commercially available MYO3B antibodies, including the HRP-conjugated version, introduces specific considerations for experimental reproducibility. Unlike monoclonal antibodies that recognize a single epitope, polyclonal antibodies bind multiple epitopes, potentially providing more robust detection but with greater lot-to-lot variability .

To enhance reproducibility when using polyclonal MYO3B antibodies:

• Lot tracking: Maintain detailed records of antibody lot numbers used in experiments

• Standardized titration: Determine optimal working dilutions for each new lot

• Internal controls: Include consistent positive and negative controls across experimental series

• Reference standards: Utilize recombinant MYO3B protein standards in quantitative assays

Researchers should recognize that polyclonal antibody performance may vary between suppliers and production batches . This variability can manifest as differences in background signal, detection sensitivity, and specificity. Establishing robust validation protocols for each new antibody lot can help mitigate these reproducibility challenges .

What are the optimal storage and handling conditions to maintain MYO3B antibody performance over time?

To maintain optimal performance of MYO3B Antibody, HRP conjugated, proper storage and handling conditions are essential. Manufacturer specifications recommend storage at -20°C or -80°C upon receipt, with particular emphasis on avoiding repeated freeze-thaw cycles that can degrade both antibody binding capacity and HRP enzymatic activity .

For long-term storage stability:

During experimental use, researchers should maintain the antibody on ice when removed from frozen storage and promptly return unused portions to appropriate storage conditions. Working dilutions should be prepared fresh for each experimental session to ensure consistent performance, particularly for quantitative applications .

What controls are essential when validating experimental results using MYO3B Antibody, HRP conjugated?

Comprehensive validation of experimental results requires implementation of multiple control types when using MYO3B Antibody, HRP conjugated. These controls address potential sources of variability and artifacts in experimental systems.

Essential controls include:

• Positive tissue controls: Samples from tissues known to express MYO3B (retina, kidney, testis) to confirm detection capability

• Negative controls:

  • Secondary antibody-only controls to assess non-specific binding

  • Samples from tissues with minimal MYO3B expression

  • Ideally, MYO3B knockout or knockdown samples when available

• Procedural controls:

  • Antigen competition assays using recombinant MYO3B protein

  • Concentration gradients to establish detection limits

  • Cross-reactivity assessments with related myosin proteins

• Technical controls:

  • Standardized protein loading controls for quantitative comparisons

  • Batch controls across multiple experimental sessions

  • Inter-laboratory validation for critical findings

Implementing these control measures provides a robust framework for distinguishing between genuine biological signals and technical artifacts, particularly important given the polyclonal nature of the available antibodies and potential cross-reactivity with other myosin family members .

How should researchers approach quantitative analysis of MYO3B expression using HRP-conjugated antibodies?

Quantitative analysis of MYO3B expression using HRP-conjugated antibodies requires systematic approaches to ensure accuracy and reproducibility. ELISA represents the primary validated application for these antibodies, making it the preferred method for quantitative assessment .

For reliable quantification:

• Standard curve development:

  • Use recombinant human MYO3B protein (1110-1276AA) as reference standard

  • Prepare fresh standard dilutions for each experimental series

  • Generate standard curves spanning physiologically relevant concentrations

• Signal optimization:

  • Determine optimal antibody dilution through titration experiments

  • Establish appropriate substrate incubation times for linear range detection

  • Validate signal-to-noise ratios across sample types

• Normalization strategies:

  • Employ appropriate housekeeping proteins as loading controls

  • Consider total protein normalization methods for tissues with variable expression

  • Account for tissue-specific matrix effects in sample preparation

• Data analysis approaches:

  • Apply appropriate curve-fitting models for standard curve interpolation

  • Conduct statistical analyses accounting for technical and biological replicates

  • Establish confidence intervals reflective of assay precision limitations

Researchers should recognize that absolute quantification requires additional validation compared to relative quantification approaches, particularly when comparing MYO3B expression across different experimental conditions or tissue types .

What are common sources of false positives/negatives when using MYO3B Antibody, HRP conjugated, and how can they be addressed?

When using MYO3B Antibody, HRP conjugated, several common issues can lead to false results. Understanding and mitigating these factors is essential for accurate data interpretation.

Sources of false positives and mitigation strategies:

• Cross-reactivity with related proteins:

  • Particularly with other myosin family members that share sequence homology

  • Mitigate by using peptide competition assays and sequence comparison analyses

  • Consider orthogonal detection methods targeting different epitopes

• Endogenous peroxidase activity:

  • Particularly problematic in tissue samples with high peroxidase content

  • Address through hydrogen peroxide quenching steps prior to antibody application

  • Validate with appropriate enzyme inhibitor controls

• Non-specific binding:

  • Can occur due to insufficient blocking or sample-specific interactions

  • Optimize blocking conditions (duration, composition, temperature)

  • Include secondary antibody-only controls in experimental design

Sources of false negatives and mitigation strategies:

• Epitope masking or destruction:

  • Can result from sample preparation methods that alter protein structure

  • Test multiple fixation and extraction protocols

  • Consider native versus denaturing conditions based on epitope characteristics

• Insufficient sensitivity:

  • May occur in tissues with low MYO3B expression

  • Implement signal amplification systems compatible with HRP

  • Optimize antibody concentration and incubation conditions

• HRP activity inhibition:

  • Can result from buffer components or sample contaminants

  • Test for inhibition using control HRP enzyme assays

  • Modify sample preparation to remove potential inhibitors

By systematically addressing these potential sources of error, researchers can substantially improve the reliability of experimental results when working with MYO3B Antibody, HRP conjugated .

How should researchers interpret conflicting results between different detection methods for MYO3B?

When researchers encounter conflicting results between different detection methods for MYO3B, a systematic approach to resolution is necessary. These discrepancies may arise from method-specific limitations rather than actual biological variations.

To address conflicting results:

• Epitope accessibility analysis:

  • Different detection methods may access different protein regions

  • Compare the epitope targeted by the HRP-conjugated antibody (amino acids 1110-1276) with epitopes targeted by other methods

  • Consider native versus denatured protein states across methods

• Method-specific sensitivity assessment:

  • Establish detection limits for each method using recombinant protein standards

  • Determine whether discrepancies occur near detection thresholds

  • Consider signal amplification for methods demonstrating lower sensitivity

• Cross-validation hierarchy:

  • Prioritize results from methods with extensive validation data

  • Consider orthogonal approaches (e.g., mass spectrometry) for definitive resolution

  • Evaluate consistency with known MYO3B expression patterns in reference tissues

• Isoform-specific detection evaluation:

  • Assess whether methods differentially detect the seven reported MYO3B isoforms

  • Design isoform-specific control experiments when possible

  • Correlate results with transcript-level analyses when available

When interpreting conflicting results, researchers should report discrepancies transparently, detailing methodological differences and proposing biological or technical explanations for observed variations. This approach advances understanding of both the protein biology and methodological limitations .

What approaches can help differentiate between specific MYO3B isoforms in experimental systems?

Differentiating between the seven reported isoforms of MYO3B presents a significant challenge that requires specialized approaches. The commercially available MYO3B Antibody, HRP conjugated targets a specific region (amino acids 1110-1276) that may be present in multiple isoforms, potentially limiting isoform specificity .

To achieve isoform-specific detection:

• Epitope mapping analysis:

  • Compare the immunogen sequence against known isoform sequences

  • Identify antibodies targeting isoform-specific regions when available

  • Design custom antibodies for unique isoform junctions or sequences

• Complementary nucleic acid approaches:

  • Implement RT-PCR with isoform-specific primers

  • Utilize RNA-Seq for comprehensive isoform profiling

  • Correlate protein-level detection with transcript isoform abundance

• Size-based discrimination:

  • Leverage molecular weight differences between isoforms in Western blot applications

  • Implement high-resolution gel systems for optimal separation

  • Consider 2D gel electrophoresis for complex samples

• Mass spectrometry validation:

  • Identify isoform-specific peptides through proteomics approaches

  • Use targeted mass spectrometry for quantitative isoform profiling

  • Correlate immunodetection results with mass spectrometry data

When absolute isoform specificity is required, researchers should consider implementing genetic approaches (e.g., isoform-specific tagging or selective knockdown) alongside immunodetection methods. This multi-modal approach provides the most comprehensive isoform characterization, particularly important when studying tissue-specific isoform expression patterns .

How might emerging antibody technologies improve MYO3B detection specificity and sensitivity?

Emerging antibody technologies offer promising approaches to enhance both specificity and sensitivity in MYO3B detection. Current polyclonal antibody limitations could be addressed through several advancing technologies.

Potential technological improvements include:

• Recombinant antibody development:

  • Single-chain variable fragments (scFvs) targeting MYO3B-specific epitopes

  • Consistent production without batch-to-batch variability of polyclonal antibodies

  • Engineered binding domains with enhanced affinity and specificity

• Nanobody/single-domain antibody applications:

  • Smaller binding domains allowing access to sterically hindered epitopes

  • Improved tissue penetration for detection in complex samples

  • Enhanced stability in various experimental conditions

• Multiparametric detection systems:

  • Multiplexed antibodies allowing simultaneous detection of multiple myosin family members

  • Co-localization studies with improved spatial resolution

  • Quantitative ratiometric approaches for expression pattern analysis

• Alternative conjugation chemistries:

  • Site-specific enzymatic labeling to preserve binding characteristics

  • Quantum dot conjugation for enhanced sensitivity and photostability

  • Click chemistry approaches for custom detection system development

These technological advances could significantly improve the research toolkit for MYO3B studies, particularly for distinguishing between closely related myosin family members and detecting low-abundance isoforms in complex biological samples .

What methodological gaps exist in current MYO3B research, and how might they be addressed?

Despite available tools for MYO3B detection, several methodological gaps limit comprehensive understanding of this protein's biological functions. Addressing these gaps requires both technical innovations and experimental design considerations.

Current methodological limitations include:

• Temporal dynamics assessment:

  • Limited tools for real-time monitoring of MYO3B activity

  • Need for methods capturing both localization and functional states

  • Potential for development of activity-based probes specific to MYO3B

• Protein interaction network characterization:

  • Incomplete understanding of MYO3B binding partners across cell types

  • Need for validated co-immunoprecipitation protocols with HRP-conjugated antibodies

  • Potential for proximity labeling approaches to identify transient interactions

• Tissue-specific function elucidation:

  • Limited characterization in physiologically relevant tissues

  • Need for improved methods for studying native MYO3B in retina, kidney, and testis

  • Potential for tissue-specific conditional knockout models

• Structural biology integration:

  • Limited correlation between antibody-based detection and structural insights

  • Need for epitope mapping in the context of protein conformation

  • Potential for structure-guided antibody development

Addressing these gaps requires interdisciplinary approaches combining traditional antibody-based detection with emerging technologies in proteomic analysis, structural biology, and functional genomics. Development of MYO3B-specific research tools would benefit from collaborative efforts between antibody developers and research groups focusing on myosin biology .