MYH11 Antibody, HRP conjugated

What is MYH11 and what is its biological significance?

MYH11 (Myosin Heavy Chain 11), also known as smooth muscle myosin heavy chain, is a critical protein that plays a fundamental role in muscle contraction and cellular movement. It functions by interacting with actin filaments to generate force, enabling various physiological processes including vasoconstriction and gastrointestinal motility . The protein exists as a hexameric assembly containing two heavy chain subunits and light chain subunits that can be either phosphorylatable or non-phosphorylatable . The phosphorylation state of myosin light chain serves as a key regulatory mechanism for smooth muscle contraction, modulated by calcium/calmodulin-dependent myosin light chain kinase . Understanding MYH11's structure and function is essential for elucidating smooth muscle physiology and pathophysiology as well as developing therapeutic strategies for conditions involving smooth muscle dysfunction .

What are the different types of MYH11 antibodies available and their applications?

Multiple types of MYH11 antibodies are available with varying host origins, clonality, and detection capabilities:

These antibodies can be obtained in both non-conjugated forms and conjugated versions with various tags including horseradish peroxidase (HRP), phycoerythrin (PE), fluorescein isothiocyanate (FITC), and multiple Alexa Fluor® conjugates .

How does HRP conjugation enhance antibody functionality in immunoassays?

HRP (horseradish peroxidase) conjugation significantly enhances antibody functionality by providing a sensitive enzymatic detection system. When HRP is conjugated to an MYH11 antibody, it catalyzes colorimetric, chemiluminescent, or fluorescent reactions in the presence of appropriate substrates, producing detectable signals. The enzymatic activity of HRP provides signal amplification, improving detection sensitivity in techniques such as Western blotting, ELISA, and immunohistochemistry . According to comparative studies, properly optimized HRP-conjugated antibodies can work at dilutions as high as 1:5000, whereas traditional methods may require more concentrated preparations working at 1:25 dilutions . This enhancement is particularly valuable when detecting low-abundance MYH11 protein in complex biological samples.

What is the optimal protocol for conjugating MYH11 antibodies with HRP?

An enhanced protocol for conjugating MYH11 antibodies with HRP incorporates a lyophilization step that significantly improves conjugation efficiency:

• HRP Activation: Oxidize carbohydrate moieties on HRP using sodium meta-periodate to generate reactive aldehyde groups

• Lyophilization: Freeze-dry the activated HRP (key modification to traditional protocols)

• Conjugation: Mix lyophilized, activated HRP with MYH11 antibody at a concentration of 1 mg/ml

• Stabilization: Reduce Schiff bases formed between HRP and antibody using sodium cyanoborohydride

• Purification: Remove unreacted components through gel filtration chromatography

• Validation: Confirm conjugation success via UV-spectroscopy and SDS-PAGE

This modified approach enables antibodies to bind more HRP molecules, resulting in conjugates with significantly enhanced sensitivity compared to traditional conjugation methods (p<0.001) .

How can researchers validate the specificity and functionality of MYH11-HRP conjugates?

A comprehensive validation approach for MYH11-HRP conjugates should include:

This multi-parameter validation ensures that the MYH11-HRP conjugate maintains both antibody specificity and enzymatic functionality.

What are the optimal working dilutions for MYH11-HRP conjugates across different applications?

Optimal working dilutions for MYH11-HRP conjugates vary by application:

When using enhanced conjugation methods incorporating lyophilization, MYH11-HRP conjugates can potentially achieve functional detection at dilutions as high as 1:5000, compared to classical conjugation methods requiring more concentrated preparations (1:25) . Each new lot of conjugate should undergo titration to determine optimal working dilutions for specific experimental conditions.

What are common challenges when using MYH11-HRP conjugates in Western blotting and how can they be addressed?

MYH11 is a high molecular weight protein (~200-250 kDa) that presents specific challenges in Western blotting:

Systematic optimization of these parameters ensures reliable detection of MYH11 using HRP-conjugated antibodies in Western blotting applications.

How can researchers minimize background and maximize signal-to-noise ratio when using MYH11-HRP conjugates?

Optimizing signal-to-noise ratio is critical for sensitive and specific detection:

• Blocking Optimization:

  • Test multiple blocking agents (BSA, casein, non-fat milk)

  • Determine optimal blocking time and temperature

  • Consider specialized blocking reagents for difficult samples

• Antibody Dilution Optimization:

  • Perform titration experiments to identify optimal concentration

  • Prepare antibody dilutions in fresh blocking buffer

  • Consider overnight incubation at 4°C for better binding kinetics

• Washing Protocol Enhancement:

  • Increase number of wash steps

  • Use appropriate detergents (Tween-20, Triton X-100)

  • Ensure adequate wash volume and agitation

• Endogenous Peroxidase Management:

  • Quench endogenous peroxidase activity with H₂O₂ pre-treatment

  • Use peroxidase blocking reagents compatible with tissue type

  • Perform control experiments without primary antibody

• Substrate Selection:

  • Match substrate sensitivity to expression level

  • Consider signal development time and stability

  • Use substrates with lower background characteristics

Implementing these strategies systematically can significantly improve detection specificity and sensitivity when working with MYH11-HRP conjugates.

How should researchers interpret contradictory results between different detection methods using MYH11-HRP conjugates?

When faced with conflicting results across different detection platforms:

• Consider Method-Specific Factors:

  • Western blot detects denatured proteins while IHC preserves native conformation

  • ELISA may detect soluble forms not visible in tissue sections

  • Flow cytometry provides single-cell resolution but may alter surface epitopes

  • Different detection methods have varying sensitivity thresholds

• Examine Antibody Characteristics:

  • Epitope accessibility may vary between applications

  • Fixation/denaturation can affect antibody binding differently

  • Clonal antibodies may recognize specific epitopes not available in all contexts

• Validation Approaches:

  • Use multiple antibodies targeting different MYH11 epitopes

  • Implement orthogonal detection methods (mRNA analysis, functional assays)

  • Include appropriate positive and negative controls for each method

  • Consider biological variables (tissue-specific expression, isoforms)

• Resolution Strategy:

  • Systematically document experimental conditions across methods

  • Standardize sample preparation when possible

  • Consider complementary approaches to resolve discrepancies

Understanding the fundamental principles and limitations of each technique is essential for proper interpretation of seemingly contradictory results.

How can MYH11-HRP conjugates be utilized for multiplex detection alongside other smooth muscle markers?

Multiplex detection strategies allow simultaneous visualization of multiple markers:

• Chromogenic Multiplex Approaches:

  • Sequential staining with different HRP substrates (DAB, AEC, TMB)

  • Use of spectral unmixing algorithms for closely related chromogens

  • Carefully designed washing steps between successive antibody applications

• Complementary Smooth Muscle Marker Panel:

  • MYH11 (contractile phenotype marker)

  • α-smooth muscle actin (broader smooth muscle marker)

  • Calponin/caldesmon (accessory contractile proteins)

  • Smoothelin (mature smooth muscle marker)

  • SM22-α/transgelin (early smooth muscle differentiation)

• Methodological Considerations:

  • Antibody stripping and reprobing between markers

  • Cross-adsorption of antibodies to prevent cross-reactivity

  • Sequential antibody application with intervening blocking steps

  • Digital analysis of multiplex staining patterns

This approach enables comprehensive phenotyping of smooth muscle cells in normal and pathological tissues with spatial context preservation.

What signal amplification strategies can enhance detection sensitivity for low-abundance MYH11?

For detecting low levels of MYH11 protein, several signal amplification methods can be employed:

The choice of amplification method should be based on the specific application, required sensitivity, and availability of equipment. Positive and negative controls are essential when using these highly sensitive detection methods.

How can researchers quantitatively analyze MYH11 expression in comparative tissue studies using HRP-conjugated antibodies?

Quantitative analysis of MYH11 expression requires standardized methodologies:

• Image Analysis for IHC/IF:

  • Standardize image acquisition parameters (exposure, gain, resolution)

  • Apply appropriate thresholding methods

  • Measure parameters such as:

    • Staining intensity (mean optical density)

    • Percent positive area

    • Integrated optical density (IOD = area × intensity)

  • Normalize to internal reference markers

• Western Blot Quantification:

  • Include protein concentration standards

  • Verify linear dynamic range of detection

  • Use appropriate loading controls

  • Apply densitometry with background subtraction

  • Normalize to housekeeping proteins or total protein stains

• ELISA-Based Quantification:

  • Generate standard curves using recombinant MYH11

  • Ensure sample preparation maintains protein integrity

  • Perform technical replicates and validate reproducibility

  • Apply appropriate curve-fitting models

• Statistical Considerations:

  • Power analysis for sample size determination

  • Appropriate statistical tests based on data distribution

  • Multiple comparison corrections

  • Correlation analysis with functional parameters

These approaches enable reliable comparative analysis of MYH11 expression across different experimental conditions or disease states with appropriate statistical rigor.

How are recent advances in antibody engineering influencing the development of next-generation MYH11-HRP conjugates?

Recent innovations in antibody engineering are transforming MYH11-HRP conjugate development:

• Recombinant Antibody Technology:

  • Recombinant rabbit monoclonal antibodies offer better specificity and sensitivity compared to traditional antibodies

  • Lot-to-lot consistency improves reproducibility of results

  • Animal origin-free formulations address ethical concerns

  • Enhanced immunoreactivity due to larger rabbit immune repertoire

• Site-Specific Conjugation Strategies:

  • Precisely controlled attachment sites prevent interference with binding regions

  • Homogeneous conjugate populations improve quantitative accuracy

  • Engineered antibody variants with incorporated conjugation tags

• Novel Linker Technologies:

  • Cleavable linkers for controlled release applications

  • Hydrophilic spacers to improve solubility and reduce aggregation

  • Branched linkers allowing attachment of multiple HRP molecules per antibody

These advances are expected to yield MYH11-HRP conjugates with improved sensitivity, specificity, and batch-to-batch consistency for research applications.

What are the emerging applications of MYH11-HRP conjugates in single-cell analysis and spatial proteomics?

MYH11-HRP conjugates are finding new applications in advanced cellular analysis:

• Single-Cell Proteomics:

  • Integration with microfluidic platforms for single-cell protein quantification

  • Coupling with mass cytometry (CyTOF) for high-dimensional analysis

  • Correlation of MYH11 expression with other cellular parameters

• Spatial Proteomics Applications:

  • Multiplexed ion beam imaging (MIBI) using metal-conjugated antibodies

  • Cyclic immunofluorescence (CycIF) for iterative staining of the same tissue

  • Co-detection by indexing (CODEX) for highly multiplexed imaging

• Emerging Integration Approaches:

  • Spatial transcriptomics correlated with MYH11 protein expression

  • Integration with single-cell RNA sequencing data

  • Multi-omics approaches combining proteomics, transcriptomics, and epigenomics

These emerging applications provide unprecedented insights into smooth muscle cell heterogeneity and function in both normal physiology and disease states.