MYL4 Antibody

What is MYL4 and what is its significance in cardiac research?

MYL4 (myosin light chain 4) encodes an essential myosin light chain that is primarily expressed in atrial tissues. It plays a critical role in atrial contractile, electrical, and structural integrity . The protein is approximately 21.6 kilodaltons in mass and is also known by alternative names including ALC1, AMLC, GT1, PRO1957, atrial myosin light chain 1, and myosin light chain 1 (embryonic muscle/atrial isoform) .

Research has demonstrated that dysfunction of MYL4 leads to heritable atrial cardiomyopathy, manifesting as progressive atrial-selective electromechanical dysfunction, tachyarrhythmias, and bradyarrhythmias requiring pacemaker implantation . This makes MYL4 an important target for researchers studying cardiac pathologies, particularly those affecting atrial function.

Which applications are commonly used with MYL4 antibodies in research?

MYL4 antibodies are versatile research tools applicable across multiple experimental techniques. According to technical specifications, MYL4 antibodies are validated for:

• Western Blot (WB): Typically used at dilutions of 1:5000-1:50000

• Immunohistochemistry (IHC): Recommended dilution of 1:150-1:600

• Immunofluorescence (IF-P): Recommended dilution of 1:200-1:800

• Flow Cytometry (FC-Intra): Using approximately 0.40 μg per 10^6 cells in a 100 μl suspension

• ELISA applications

Importantly, these applications allow researchers to detect and quantify MYL4 expression in various experimental systems, including tissues and cell cultures, with antibodies showing reactivity across multiple species including human, mouse, rat, and pig samples .

How should tissue processing be optimized for MYL4 detection using immunohistochemistry?

For optimal MYL4 detection in immunohistochemistry applications, tissue processing should follow specific protocols. Antigen retrieval is a critical step, with recommended procedures including:

• Primary recommendation: TE buffer at pH 9.0

• Alternative method: Citrate buffer at pH 6.0

Human heart tissue has been validated for positive IHC detection of MYL4, making it an appropriate positive control when establishing the protocol . The recommended antibody dilution for IHC applications is 1:150-1:600, though researchers should perform titration experiments in their specific testing systems to determine optimal concentrations .

For preservation of MYL4 antibody reagents, store at -20°C in the provided buffer (typically PBS with 0.02% sodium azide and 50% glycerol at pH 7.3). Most commercial preparations remain stable for one year after shipment when properly stored .

How do MYL4 expression levels correlate with different types of atrial fibrillation, and what are the methodological considerations for such analyses?

Research examining the relationship between MYL4 expression and atrial fibrillation (AF) has revealed significant correlations that vary by AF subtype. Studies have demonstrated that MYL4 levels show a progressive decrease that correlates with the chronicity and severity of AF, as shown in the following data:

Statistical analysis showed significant differences among these groups (F=56.27, P<0.001) .

When designing studies to investigate such correlations, researchers should consider:

• Precise phenotyping of AF patients according to established clinical classifications

• Standardization of sample collection timing relative to AF episodes

• Use of matched controls to account for age, sex, and comorbidities

• Employment of multiple detection methods for MYL4 (protein and mRNA levels)

• Correlation with other biomarkers such as miR-106, which has been found to be inversely correlated with MYL4 levels in AF patients

What methodological approaches can be used to study the functional consequences of MYL4 mutations in cardiac tissue?

Investigating the functional impact of MYL4 mutations requires sophisticated methodological approaches. Based on published research, the following complementary strategies have proven effective:

• Genetic animal models:

  • Gene knock-in models, such as MYL4 p.E11K knock-in rats, which recapitulate the human phenotype

  • Complete knockout models to study loss-of-function effects

  • Selective deletion models with spatial or temporal control

• Cell-based approaches:

  • Adenoviral transfer of mutant MYL4 genes to neonatal cardiomyocytes

  • Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining to assess apoptotic cell death

  • Assessment of proapoptotic and profibrotic signaling pathways

• Electrophysiological assessment:

  • In vivo electrocardiography to document conduction abnormalities

  • Ex vivo optical mapping to detect abnormal conduction patterns

  • Patch clamp studies of ionic currents in isolated atrial myocytes

• Structural analysis:

  • Histological assessment of fibrosis using Masson's trichrome staining

  • Electron microscopy to evaluate sarcomeric organization

  • Immunohistochemistry to detect changes in the distribution of gap junction proteins

These methodological approaches should be selected based on the specific research question and integrated to provide comprehensive understanding of how MYL4 mutations affect cardiac structure and function.

How can researchers troubleshoot inconsistent MYL4 antibody performance across different experimental systems?

Inconsistent antibody performance is a common challenge in research. For MYL4 antibodies specifically, consider these methodological approaches to troubleshooting:

• Validation with appropriate controls:

  • Positive controls: Use tissues with confirmed high MYL4 expression (pig heart tissue, rat heart tissues, mouse heart tissues)

  • Negative controls: Use MYL4 knockout samples when available, or tissues known to lack MYL4 expression

  • Antibody controls: Include isotype controls and secondary-only controls

• Optimization of antibody concentration:

  • Perform titration experiments across a wide concentration range

  • Western Blot applications may require significantly different dilutions (1:5000-1:50000) compared to IHC (1:150-1:600)

  • Individual antibody lots may require optimization

• Species cross-reactivity verification:

  • Confirm antibody reactivity with your species of interest

  • MYL4 antibodies have demonstrated reactivity with human, mouse, rat, and pig samples, but performance may vary

  • Consider epitope conservation when selecting antibodies for non-validated species

• Sample preparation considerations:

  • For IHC applications, compare antigen retrieval methods (TE buffer pH 9.0 vs. citrate buffer pH 6.0)

  • For Western Blot, optimize protein extraction buffers to ensure complete solubilization

  • Be aware that the observed molecular weight of MYL4 is typically 22-25 kDa

How can MYL4 antibodies be used to investigate the relationship between atrial fibrillation and MYL4 expression?

MYL4 antibodies provide valuable tools for investigating the correlation between atrial fibrillation and MYL4 expression through multiple methodological approaches:

• Clinical sample analysis:

  • Immunohistochemical staining of atrial tissue biopsies to compare MYL4 expression between AF patients and controls

  • Quantitative Western blot analysis of atrial tissue samples to measure MYL4 protein levels

  • Correlation of MYL4 expression with clinical variables and AF classification

• Combined biomarker approach:

  • Integration of MYL4 protein detection with measurement of other biomarkers like miR-106

  • As shown in research, MYL4 levels (0.24 ± 0.08) are significantly reduced in AF patients compared to controls (1.55 ± 0.79), while miR-106 shows an inverse relationship

• Longitudinal studies:

  • Serial measurements of MYL4 in patients progressing from paroxysmal to persistent AF

  • Correlation with electrophysiological parameters and structural remodeling assessed by imaging

• Therapeutic response monitoring:

  • Assessment of MYL4 expression changes following antiarrhythmic therapy or ablation procedures

  • Potential use as a prognostic marker for treatment response

This integrative approach allows researchers to establish not only correlative relationships but also explore potential mechanistic links between MYL4 dysfunction and atrial fibrillation pathogenesis.

What are the technical considerations when using MYL4 antibodies in flow cytometry applications?

Flow cytometry applications using MYL4 antibodies require specific technical considerations to ensure reliable results:

• Cell preparation and fixation:

  • Optimization of permeabilization protocols is critical since MYL4 is an intracellular protein

  • Fixation methods may affect epitope accessibility and should be carefully selected

  • When analyzing cardiac cells, protocols must account for the large size and complex structure of cardiomyocytes

• Antibody concentration and staining:

  • For flow cytometry applications, the recommended starting concentration is 0.40 μg per 10^6 cells in a 100 μl suspension

  • Titration experiments should be performed to determine optimal antibody concentrations

  • Include appropriate isotype controls to establish specific binding

• Validated cell types:

  • C2C12 cells have been validated for positive intracellular flow cytometry detection of MYL4

  • When analyzing primary cardiac cells, consider potential autofluorescence issues

  • For cardiomyocytes derived from induced pluripotent stem cells (iPSCs), confirm MYL4 expression patterns during differentiation

• Data analysis considerations:

  • Use appropriate gating strategies to distinguish MYL4-positive populations

  • Consider co-staining with cardiomyocyte markers to identify specific cell populations

  • When comparing samples, ensure consistent instrument settings and calibration

These technical considerations help ensure that flow cytometry data using MYL4 antibodies is robust and reproducible across different experimental settings.

How is MYL4 expression being studied in the context of cardiac regeneration and repair?

MYL4 expression patterns provide important insights into cardiac regeneration and repair processes. Research has indicated that:

• Embryonic to adult transition patterns:

  • MYL4 is typically expressed in embryonic ventricles but is downregulated postnatally

  • In adult hearts, MYL4 expression is normally restricted to atrial tissue

  • During pathological conditions, ventricular reexpression of MYL4 may occur

• Regenerative medicine applications:

  • MYL4 antibodies can be used to monitor cardiomyocyte maturation in stem cell-derived cardiac tissues

  • Expression patterns help distinguish between immature/fetal-like and mature cardiomyocyte phenotypes

  • Changes in MYL4 expression may serve as markers of successful cardiac regeneration

• Response to injury:

  • Studies suggest that reactivation of MYL4 expression in ventricular myocytes following injury may be beneficial for improving cardiac function

  • MYL4 antibodies can track this reexpression in experimental models of myocardial infarction or heart failure

  • Temporal patterns of expression provide insights into repair mechanisms

• Therapeutic targeting potential:

  • Understanding the functional consequences of MYL4 expression in regenerating cardiac tissue could inform regenerative medicine approaches

  • MYL4 could potentially serve as a therapeutic target to enhance cardiac repair processes

These research directions highlight the importance of MYL4 antibodies as tools for studying cardiac development, injury response, and regenerative potential.

What are the methodological considerations for using MYL4 antibodies in high-throughput screening applications?

Adaptation of MYL4 antibody-based assays for high-throughput screening requires specific methodological considerations:

• Assay miniaturization and optimization:

  • Development of ELISA-based methods with optimized antibody concentrations

  • Validation of signal-to-background ratios across plate positions

  • Establishment of appropriate positive and negative controls for Z-factor calculation

• Automated image analysis for high-content screening:

  • When using immunofluorescence-based detection, develop algorithms for automated quantification of MYL4 expression

  • For IHC applications, consider digital pathology approaches with appropriate training of analysis algorithms

  • Account for subcellular localization patterns specific to MYL4

• Quality control measures:

  • Include technical replicates across plates to assess inter-plate variability

  • Monitor antibody performance across different lots

  • Use validated samples with known MYL4 expression levels as internal standards

• Data normalization and analysis considerations:

  • Develop normalization strategies to account for well-to-well variations

  • Consider statistical approaches appropriate for high-dimensional data

  • Integrate MYL4 expression data with other measured parameters for comprehensive analysis

These methodological considerations support the adaptation of MYL4 antibody applications from standard laboratory techniques to high-throughput screening platforms suitable for drug discovery or large-scale genetic studies.

How should researchers approach the selection of appropriate MYL4 antibodies for specific applications?

Researchers should adopt a systematic approach when selecting MYL4 antibodies, considering several key factors:

• Experimental application alignment:

  • Match antibody validation data with intended application (WB, IHC, IF, FC)

  • Review published literature for successful application of specific antibody clones

  • Consider whether monoclonal or polyclonal antibodies are more appropriate for your specific research question

• Species reactivity requirements:

  • Verify validated reactivity with your experimental model species (human, mouse, rat, pig)

  • If working with non-validated species, analyze epitope conservation across species

  • Consider using multiple antibodies recognizing different epitopes for confirmation

• Technical specifications evaluation:

  • Review molecular weight information (MYL4 is typically observed at 22-25 kDa)

  • Check recommended dilution ranges for your application

  • Assess storage requirements and stability information

• Validation strategy development:

  • Plan appropriate positive controls (pig heart tissue, K-562 cells, Mo7e cells, TF-1 cells, rat heart tissues, mouse heart tissues)

  • Consider including negative controls through genetic approaches (knockdown/knockout) when possible

  • Design experiments to confirm specificity through multiple methodological approaches

This systematic approach enhances the likelihood of selecting appropriate MYL4 antibodies that will generate reliable and reproducible results across various experimental systems, advancing our understanding of MYL4 biology in cardiac health and disease.