MYL3 Antibody

What is MYL3 and what is its biological significance in muscle physiology?

MYL3 (Myosin Light Chain 3) is an alkali light chain of myosin also referred to as ventricular isoform (MLC1v) and slow skeletal muscle isoform. It functions as a regulatory light chain that activates the mechanical movement of myosin chains, significantly impacting cardiac and skeletal muscle contraction. MYL3 interacts with proteins such as tropomyosin and troponin which are crucial for regulating muscle contraction . The protein does not bind calcium directly but plays an essential role in the proper functioning of cardiac and skeletal muscles through its phosphorylation state, which is critical for regulating muscle contraction . Notably, mutations in MYL3 have been identified as a cause of mid-left ventricular chamber type hypertrophic cardiomyopathy .

What is the molecular structure and evolutionary conservation of MYL3?

MYL3 has a calculated molecular weight of approximately 22 kDa, though observed molecular weights in laboratory testing often range between 22-27 kDa depending on post-translational modifications and experimental conditions . The human MYL3 protein shows high sequence identity (91%) with mouse MYL3, underscoring its strong evolutionary conservation . This conservation suggests its fundamental importance in muscle physiology across mammalian species. The structure of myosin, including MYL3, consists of a long asymmetric molecule featuring a globular head and a long tail, which facilitates interaction with actin filaments during muscle contraction .

What are the typical applications for MYL3 antibodies in research?

MYL3 antibodies are versatile tools employed in multiple experimental methodologies:

These applications have been validated across human, mouse, rat, and pig samples, making MYL3 antibodies valuable for comparative studies .

How should researchers select the appropriate MYL3 antibody for specific experimental applications?

Selection of an appropriate MYL3 antibody requires consideration of multiple factors:

• Target species reactivity: Verify cross-reactivity with your experimental model. Many MYL3 antibodies demonstrate reactivity with human, mouse, and rat samples, but specificity varies between products .

• Application compatibility: Some antibodies perform better in certain applications. For example, monoclonal antibody 66286-1-Ig shows exceptional performance in Western blotting with dilutions up to 1:50000, while polyclonal antibody 10913-1-AP demonstrates broader application versatility across WB, IHC, IF/ICC, FC, and IP .

• Epitope recognition: Consider which region of MYL3 your antibody recognizes. Antibodies targeting different epitopes (e.g., N-terminal vs. internal regions) may yield different results depending on protein conformation and post-translational modifications .

• Clone type: Monoclonal antibodies offer higher specificity and reproducibility, while polyclonal antibodies may provide higher sensitivity and recognition of multiple epitopes .

• Validation data: Review published literature and manufacturer validation data to confirm the antibody's performance in your specific application .

What controls should be included when using MYL3 antibodies in experimental protocols?

Rigorous experimental design with appropriate controls is essential:

• Positive tissue controls: Include samples known to express MYL3 such as heart tissue, ventricular muscle, or slow skeletal muscle for validation .

• Negative controls: Include tissues that do not express MYL3 or use secondary antibody-only controls to assess non-specific binding.

• Blocking peptide controls: When available, use the immunizing peptide to compete with antibody binding and demonstrate specificity.

• Loading controls: For Western blots, include housekeeping proteins (e.g., GAPDH, β-actin) to normalize MYL3 expression.

• Isotype controls: For flow cytometry or immunohistochemistry, use isotype-matched irrelevant antibodies to assess background staining.

• Species cross-reactivity validation: If working with non-human samples, verify antibody performance in your specific species of interest .

What are the optimal sample preparation methods for MYL3 antibody applications?

Sample preparation significantly impacts experimental success:

For Western Blotting:

• Extract proteins using RIPA or NP-40 buffers containing protease inhibitors

• Include phosphatase inhibitors if studying phosphorylation states

• Optimal protein loading typically ranges from 20-50 μg per lane

• Denature samples at 95-100°C for 5 minutes in reducing sample buffer

For Immunohistochemistry:

• For formalin-fixed, paraffin-embedded samples, antigen retrieval with TE buffer pH 9.0 is recommended

• Alternative antigen retrieval with citrate buffer pH 6.0 may be performed if needed

• 4-10 μm section thickness is optimal for visualization

For Immunofluorescence:

• Fixation with 4% paraformaldehyde for 10-15 minutes at room temperature

• Permeabilization with 0.1-0.5% Triton X-100 for intracellular proteins

How can researchers optimize Western blotting protocols for MYL3 detection?

Western blotting for MYL3 can be optimized with these technical considerations:

• Gel percentage selection: Use 12-15% acrylamide gels for optimal resolution of MYL3 (22 kDa)

• Transfer conditions: Employ semi-dry transfer at 15-20V for 30-45 minutes or wet transfer at 100V for 1 hour with methanol-containing transfer buffer to improve transfer of low molecular weight proteins

• Blocking optimization: 5% non-fat dry milk in TBST is generally effective, though 3-5% BSA may reduce background for some antibodies

• Primary antibody incubation:

  • Dilution ranges from 1:5000 to 1:50000 depending on the specific antibody

  • Incubate overnight at 4°C for optimal sensitivity

  • For rabbit polyclonal antibodies, a 1:500-1:2000 dilution is typically effective

• Signal development: Enhanced chemiluminescence (ECL) with longer exposure times may be necessary for detecting low abundance MYL3

• Expected band patterns: Anticipate bands at 22-27 kDa, with potential variation based on post-translational modifications

What methodological approaches enhance immunohistochemical detection of MYL3 in tissue sections?

For optimal IHC results with MYL3 antibodies:

• Antigen retrieval optimization:

  • Heat-induced epitope retrieval using TE buffer pH 9.0 is generally recommended

  • For challenging samples, experiment with alternative retrieval methods such as citrate buffer pH 6.0

• Antibody dilution ranges:

  • For polyclonal antibodies: 1:500-1:2000

  • For monoclonal antibodies: 1:20-1:200

  • Titration experiments are recommended to determine optimal concentration

• Detection systems:

  • DAB (3,3'-diaminobenzidine) chromogen provides reliable visualization

  • For dual staining experiments, alkaline phosphatase-based detection can be used as a secondary detection method

• Counterstaining:

  • Light hematoxylin counterstaining provides optimal nuclear contrast

  • Avoid overstaining which can mask weak MYL3 signals

• Tissue considerations:

  • Human cardiac and skeletal muscle tissues serve as excellent positive controls

  • Staining patterns should show cytoplasmic distribution in muscle fibers

What troubleshooting strategies address common challenges with MYL3 antibodies?

How do MYL3 expression patterns differ across cardiac and skeletal muscle types?

MYL3 demonstrates tissue-specific expression patterns that inform understanding of muscle physiology:

• Cardiac expression: Predominantly expressed in ventricular myocardium as the ventricular isoform (MLC1v)

• Skeletal muscle expression: Primarily found in slow skeletal muscle fibers (type I fibers)

• Expression comparison:

  • Highest expression levels are typically observed in heart tissue

  • Moderate expression in slow skeletal muscle

  • Minimal or absent expression in fast skeletal muscle

• Developmental regulation: Expression patterns change during development and in response to physiological or pathological conditions

• Pathological alterations: Changes in MYL3 expression or phosphorylation state occur in various cardiac diseases, particularly hypertrophic cardiomyopathy

How can MYL3 antibodies be utilized in cardiovascular disease research?

MYL3 antibodies provide valuable tools for cardiovascular research:

• Hypertrophic cardiomyopathy (HCM) studies:

  • Detect altered expression or localization of MYL3 in HCM patient samples

  • Evaluate the effects of MYL3 mutations on protein stability and function

  • Mutations in MYL3 have been identified as causes of mid-left ventricular chamber type HCM

• Cardiac development research:

  • Track MYL3 expression during cardiomyocyte differentiation

  • Examine isoform switching during heart development

• Cardiac injury models:

  • Monitor MYL3 release as a potential biomarker of cardiac damage

  • Study MYL3 as a target for caspase-3 in dying cardiomyocytes

• Therapeutic development:

  • Evaluate the effects of experimental drugs or interventions on MYL3 expression or function

  • MYL3 has been identified as a potential serum biomarker for drug-induced cardiac toxicity

What considerations are important when analyzing MYL3 phosphorylation states?

Analysis of MYL3 phosphorylation requires specific methodological approaches:

• Phosphorylation significance:

  • Phosphorylation state of MYL3 is critical for regulating muscle contraction

  • Changes in phosphorylation occur during normal physiological processes and in disease states

• Sample preparation:

  • Include phosphatase inhibitors in all buffers during sample preparation

  • Avoid freeze-thaw cycles that may affect phosphorylation status

  • Consider using phosphorylation-specific antibodies when available

• Analytical techniques:

  • Phos-tag SDS-PAGE can resolve phosphorylated from non-phosphorylated forms

  • 2D gel electrophoresis separates phosphorylated isoforms based on charge differences

  • Mass spectrometry provides precise identification of phosphorylation sites

• Controls and standards:

  • Include both phosphatase-treated and untreated samples as controls

  • Use positive controls with known phosphorylation states

• Quantification approaches:

  • Densitometric analysis of Western blots with phospho-specific antibodies

  • Ratio analysis of phosphorylated to total MYL3 provides normalization

How are MYL3 antibodies being used in stem cell and regenerative medicine research?

MYL3 antibodies have emerging applications in stem cell research:

• Cardiomyocyte differentiation monitoring:

  • MYL3 serves as a terminal differentiation marker for ventricular cardiomyocytes

  • Antibodies enable tracking of differentiation efficiency in stem cell protocols

• Engineered heart tissue assessment:

  • Evaluating MYL3 expression and localization in tissue-engineered cardiac constructs

  • Comparing engineered tissues to native myocardium

• Single-cell analysis:

  • Flow cytometry with MYL3 antibodies enables sorting of cardiomyocyte populations

  • Immunofluorescence allows evaluation of cellular heterogeneity in differentiated cultures

• Functional maturation studies:

  • Correlation of MYL3 expression patterns with functional parameters in developing cardiomyocytes

  • Assessment of isoform switching during maturation

What methodological approaches can identify MYL3 protein-protein interactions?

Multiple techniques leveraging MYL3 antibodies can elucidate protein interaction networks:

• Co-immunoprecipitation (Co-IP):

  • Use MYL3 antibodies to pull down MYL3 and associated proteins

  • Western blotting for suspected interaction partners

  • Recommended antibody amounts: 0.5-4.0 μg for 1.0-3.0 mg of total protein lysate

• Proximity ligation assay (PLA):

  • Detect in situ protein-protein interactions with spatial resolution

  • Requires MYL3 antibody and antibody against suspected interaction partner

• Immunofluorescence co-localization:

  • Double-labeling with MYL3 and partner protein antibodies

  • Confocal microscopy analysis of spatial overlap

  • Recommended antibody dilutions: 1:50-1:500

• Cross-linking coupled with immunoprecipitation:

  • Chemical cross-linking preserves transient interactions

  • MYL3 antibodies pull down cross-linked complexes

• Mass spectrometry following immunoprecipitation:

  • Unbiased identification of MYL3 interaction partners

  • Quantitative approaches can compare interactions under different conditions

How can researchers optimize detection of MYL3 mutations associated with cardiomyopathies?

Detecting MYL3 mutations requires integrated methodological approaches:

• Antibody-based detection of mutant proteins:

  • Wild-type specific antibodies may show reduced binding to mutant forms

  • Comparing staining patterns between wild-type and mutant samples

• Expression level analysis:

  • Quantitative Western blotting to assess potential changes in protein stability

  • qPCR validation of transcriptional effects

• Subcellular localization studies:

  • Immunofluorescence to detect altered localization of mutant proteins

  • Co-staining with sarcomeric markers to assess incorporation into myofibrils

• Functional impact assessment:

  • Phosphorylation state analysis of mutant proteins

  • Correlation with functional parameters in cardiomyocyte models

• Patient-derived samples:

  • Comparing antibody staining patterns in biopsy samples from patients with MYL3 mutations

  • Validating findings in engineered cell models expressing specific mutations

What quality control measures ensure reliable results with MYL3 antibodies?

Implementation of rigorous quality control enhances experimental reliability:

• Antibody validation checklist:

  • Confirm target specificity through knockout/knockdown controls

  • Verify recognition of recombinant MYL3 protein

  • Test cross-reactivity with closely related proteins (e.g., other myosin light chains)

  • Compare results with multiple antibodies recognizing different epitopes

• Batch testing protocols:

  • Always test new antibody lots against previous lots

  • Maintain reference samples for consistency checks

  • Document lot-specific optimal dilutions

• Storage and handling:

  • Store antibodies at -20°C in small aliquots to avoid freeze-thaw cycles

  • Keep detailed records of antibody performance over time

  • Follow manufacturer recommendations for buffer conditions

• Application-specific validation:

  • Validate each antibody independently for each application

  • Establish clear acceptance criteria for each experimental system

What emerging technologies are enhancing MYL3 antibody applications?

Technological advances are expanding the utility of MYL3 antibodies:

• Super-resolution microscopy:

  • Nanoscale visualization of MYL3 integration within sarcomeric structures

  • Improved resolution of structural changes in disease models

• In vivo imaging approaches:

  • Fluorescently-labeled MYL3 antibody fragments for live imaging

  • Non-invasive monitoring of cardiac damage markers

• Multiplexed detection systems:

  • Simultaneous visualization of multiple sarcomeric proteins

  • Mass cytometry for single-cell protein profiling

• Antibody engineering:

  • Recombinant antibody fragments with improved tissue penetration

  • Bi-specific antibodies for novel detection approaches

• AI-assisted image analysis:

  • Automated quantification of staining patterns

  • Machine learning algorithms for pattern recognition in complex tissues