MYL4 Antibody

What types of MYL4 antibodies are available for research applications?

Several types of MYL4 antibodies are available for research purposes, varying in host organisms, clonality, and target epitopes. The following table summarizes key commercial antibodies:

These antibodies provide researchers with multiple options for detecting MYL4 in various experimental contexts .

What are the recommended storage conditions for MYL4 antibodies?

For optimal antibody performance and longevity, MYL4 antibodies should be stored at -20°C as received . Most commercially available antibodies are prepared in buffer solutions containing stabilizers and preservatives, such as:

• PBS (pH 7.3)

• 1% BSA

• 50% glycerol

• 0.02% sodium azide

These components help maintain antibody integrity during storage. Avoid repeated freeze-thaw cycles by aliquoting the antibody upon first thaw. For working solutions, store at 4°C for up to one month. Always refer to manufacturer-specific recommendations as storage conditions may vary between products.

What are the optimal conditions for Western blotting with MYL4 antibodies?

Achieving reliable and reproducible Western blot results with MYL4 antibodies requires optimization of several parameters:

Sample Preparation:

• Use RIPA buffer supplemented with protease inhibitors for tissue/cell lysis

• Load 20-50 μg of total protein per lane

• Use 12-15% SDS-PAGE gels (recommended due to MYL4's size of 21.565 kDa)

Antibody Conditions:

• Primary antibody dilutions:

  • Mouse monoclonal (OTI1H6): 1:2000

  • Other MYL4 antibodies: typically 1:500-1:1000

• Incubation: Overnight at 4°C

• Secondary antibody: Anti-mouse/rabbit HRP at 1:15,000

• Incubation: 2 hours at room temperature

Controls:

• Positive control: HEK293T cells transfected with MYL4 expression vectors

• Loading control: Anti-β-actin (1:10,000)

Following these conditions will help ensure specific detection of MYL4 protein with minimal background.

How can I optimize immunohistochemistry protocols for MYL4 detection?

Successful immunohistochemical detection of MYL4 requires attention to tissue preparation, antigen retrieval, and antibody conditions:

Tissue Preparation:

• Fix tissues in 10% neutral buffered formalin

• Paraffin embedding with standard processing protocols

• Section tissues at 4-6 μm thickness

Antigen Retrieval:

• Heat-induced epitope retrieval in citrate buffer (pH 6.0)

• Boil for 15-20 minutes followed by cooling to room temperature

Antibody Conditions:

• Blocking: 5-10% normal serum (matching secondary antibody species)

• Primary antibody: Anti-MYL4 monoclonal (e.g., M08496) at 1:150 dilution

• Incubation: Overnight at 4°C

• Secondary antibody: HRP-conjugated or fluorescently labeled

• Detection: DAB substrate for brightfield or fluorescence imaging

Validation:

• Include positive control tissue (atrial tissue)

• Include negative control (omitting primary antibody)

• Compare with known MYL4 expression patterns

This protocol can be adapted for frozen sections with appropriate modifications to fixation and permeabilization steps.

What immunofluorescence methods work best for studying MYL4 in cultured cells?

For immunofluorescence studies of MYL4 in cultured cells:

Cell Preparation:

• Grow cells on glass coverslips coated with appropriate substrate

• Fix with 4% paraformaldehyde for 15 minutes at room temperature

• Permeabilize with 0.2% Triton X-100 for 10 minutes

Staining Protocol:

• Block in 5% BSA for 1 hour at room temperature

• Incubate with anti-MYL4 antibody (e.g., 67533-1-Ig at 1:500) overnight at 4°C

• Wash 3× with PBS

• Incubate with fluorescent secondary antibody (e.g., DyLight 488 goat anti-mouse IgG at 1:1000) for 2 hours

• Counterstain nuclei with DAPI (1:100) for 10 minutes

• Mount with anti-fade mounting medium

Imaging:

• Capture images using fluorescence microscopy (e.g., Axio Vert A1, ZEISS)

• Use appropriate filter sets for selected fluorophores

• Analyze using quantitative image analysis software

This method has been successfully used to visualize MYL4 in myogenic differentiation studies .

How can MYL4 antibodies be used to study atrial cardiomyopathy mechanisms?

MYL4 antibodies serve as valuable tools for investigating molecular mechanisms underlying atrial cardiomyopathy:

Expression Analysis:

• Western blotting to quantify MYL4 protein levels in:

  • Patient-derived samples

  • Animal models with MYL4 mutations (e.g., p.E11K knock-in rats)

  • Control vs. disease states

Structural Studies:

• Immunohistochemistry to assess:

  • MYL4 localization in atrial tissue

  • Structural remodeling

  • Correlation with fibrosis markers

Autophagy Investigation:

• Recent research has demonstrated that MYL4 regulates autophagic flux in atrial cardiomyocytes

• Co-immunostaining of MYL4 with autophagy markers to visualize:

  • Autophagosome formation (LC3-II)

  • Autophagic flux (p62/SQSTM1)

  • Lysosomal positioning and acidification

Therapeutic Assessment:

• Monitoring MYL4 expression following interventions:

  • Adenoviral gene transfer of wild-type MYL4

  • Pharmacological modulators of autophagy

  • Evaluation of structural and functional recovery

These approaches have helped establish that MYL4 overexpression can attenuate atrial structural remodeling and autophagy dysfunction in experimental models .

What methods can detect functional consequences of MYL4 mutations?

MYL4 mutations, particularly p.E11K, have been associated with heritable atrial cardiomyopathy. Several methodological approaches can characterize these functional changes:

Genetic Analysis:

• Exome sequencing to identify variants (e.g., c.31G>A [p.E11K])

• Segregation analysis in family pedigrees (logarithm of odds score >5.3 for atrial standstill)

Animal Models:

• Comparison between:

  • MYL4 p.E11K knock-in rats

  • MYL4 knockout rats

  • Control rats with adjacent 4-amino-acid deletion (showing no phenotype)

Functional Assessments:

• Electrophysiological studies:

  • ECG for arrhythmia detection

  • Assessment of atrial standstill

• Echocardiography for structural and contractile evaluation

Molecular Characterization:

• Electron microscopy to visualize:

  • Sarcomere organization

  • Accumulation of undegraded autophagic vesicles

• Immunostaining to assess:

  • Lysosomal positioning and mobility

  • Lysosomal acidification and maturation

These comprehensive methods have established that both MYL4 mutation and absence lead to similar atrial cardiomyopathy phenotypes, highlighting the protein's essential role in atrial function .

How do I troubleshoot non-specific binding with MYL4 antibodies?

When encountering non-specific binding with MYL4 antibodies, consider these troubleshooting approaches:

Antibody Validation:

• Verify antibody specificity using:

  • Positive controls (HEK293T cells transfected with MYL4)

  • Negative controls (non-transfected cells)

  • Protein source matching (human, mouse, rat) with antibody reactivity

Blocking Optimization:

• Increase blocking time (2-3 hours)

• Try alternative blocking agents:

  • 5% non-fat dry milk

  • 5% BSA

  • Commercial blocking solutions

Antibody Dilution:

• Test multiple primary antibody dilutions (e.g., 1:500, 1:1000, 1:2000)

• Optimize secondary antibody concentration

• Reduce incubation time if overdevelopment occurs

Wash Conditions:

• Increase wash duration and number (5× washes, 5 minutes each)

• Add detergent (0.1-0.3% Tween-20) to wash buffer

• Use gentle agitation during washes

Cross-Adsorption:

• Pre-adsorb primary antibody with tissue lysate from negative control samples

• Use species-specific blocking peptides when available

These systematic approaches can help identify and eliminate sources of non-specific binding for cleaner, more interpretable results.

How does MYL4 contribute to atrial fibrillation pathophysiology?

Research has revealed important connections between MYL4 dysfunction and atrial fibrillation (AF):

Structural Basis:

• MYL4 is essential for atrial electrical, functional, and structural integrity

• MYL4 dysfunction leads to progressive atrial remodeling and fibrosis

• Pathogenic mutations (e.g., p.E11K) result in heritable atrial cardiomyopathy with various arrhythmias

Molecular Mechanisms:

• Autophagy dysregulation:

  • MYL4 mutations impair autophagy flux both in vitro and in vivo

  • Undegraded autophagic vesicles accumulate in mutant atrial tissue

  • Lysosomal positioning and mobility are regulated by MYL4

Experimental Evidence:

• Animal models with MYL4 mutations demonstrate:

  • Progressive atrial fibrosis

  • Electromechanical dysfunction

  • Tachyarrhythmias and bradyarrhythmias requiring pacemaker implantation

Therapeutic Implications:

• MYL4 overexpression via adenoviral gene transfer attenuates:

  • Atrial structural remodeling

  • Autophagy dysfunction

  • Fibrotic changes

This research suggests that targeting the MYL4-autophagy axis could provide novel therapeutic strategies for treating atrial fibrillation, particularly in cases with genetic predisposition.

What is the role of MYL4 in skeletal muscle development and disease?

While MYL4 is primarily known for its cardiac function, research has uncovered important roles in skeletal muscle:

Developmental Expression:

• MYL4 shows specific expression patterns during myogenic differentiation

• Expression can be tracked at different time points (0, 2, 4, 6, 8 days) during myogenesis

Functional Studies:

• siRNA-mediated knockdown of MYL4 affects myotube formation

• Differentiation index (nuclei in myotubes to total nuclei) changes with MYL4 manipulation

Species-Specific Patterns:

• MYL4 distribution varies between species and breeds (e.g., Ningxiang pigs vs. Large White pigs)

• Different isoforms may be identified through 3′ RACE and sequencing

Methodological Approaches:

• Western blotting with anti-MYL4 antibodies to quantify expression changes

• Immunofluorescence to visualize MYL4 localization during differentiation

• Co-staining with myogenic markers (MyoD, MyoG, MyHC) to correlate with differentiation stages

These findings suggest that MYL4 may have broader roles in muscle biology beyond its cardiac functions, with potential implications for skeletal muscle development and disease.

What are emerging applications of MYL4 antibodies in precision medicine?

MYL4 antibodies show promising potential in precision medicine approaches:

Diagnostic Applications:

• Identification of MYL4-related cardiomyopathies:

  • Immunohistochemical analysis of endomyocardial biopsies

  • Detection of aberrant MYL4 expression or localization

  • Correlation with genetic testing results

Patient Stratification:

• Classifying patients based on MYL4 expression patterns

• Identifying responders to autophagy-modulating therapies

• Personalizing treatment plans based on molecular profiles

Therapeutic Monitoring:

• Assessing efficacy of gene therapy approaches

• Quantifying MYL4 restoration following interventions

• Correlating protein levels with functional improvements

Biomarker Development:

• Evaluating MYL4 as a potential biomarker for:

  • Early detection of atrial dysfunction

  • Disease progression monitoring

  • Treatment response assessment

As research progresses, MYL4 antibodies may become valuable tools in clinical decision-making for patients with atrial cardiomyopathies and related disorders.

How can emerging technologies enhance MYL4 research?

Several cutting-edge technologies can advance MYL4 research:

Single-Cell Analysis:

• Single-cell RNA sequencing to identify cell type-specific MYL4 expression patterns

• Single-cell proteomics to detect protein-level changes

• Spatial transcriptomics to map MYL4 expression in tissue context

Advanced Imaging:

• Super-resolution microscopy for nanoscale visualization of MYL4 localization

• Live-cell imaging to track lysosomal dynamics regulated by MYL4

• FRET-based approaches to study protein-protein interactions

Genome Editing:

• CRISPR/Cas9 to introduce specific MYL4 mutations

• Base editing for precise correction of pathogenic variants

• Generation of isogenic cell lines for controlled comparisons

Computational Approaches:

• Molecular dynamics simulations to predict mutation effects

• Machine learning to identify patterns in large datasets

• Network analysis to place MYL4 in broader signaling contexts

Organ-on-Chip Technologies:

• Miniaturized heart models incorporating MYL4 mutations

• Multi-cellular systems to study cell-cell interactions

• Testing therapeutic interventions in human-relevant models

These technologies can provide deeper insights into MYL4 function and potentially accelerate the development of targeted therapies for MYL4-related diseases.