MYL2 Antibody

What is MYL2 and why is it an important research target?

MYL2 (Myosin Light Chain 2) is a contractile protein with critical functions in heart development and cardiac muscle contraction. In humans, the canonical protein consists of 166 amino acid residues with a molecular mass of approximately 18.8 kDa. MYL2 is primarily localized in the cytoplasm and is highly expressed in type I muscle fibers .

This protein is also known by several synonyms including MFM12, MLC-2, MLC-2s/v, MLC-2v, MLC2, and CMH10. As a regulatory component of the myosin complex, MYL2 plays an essential role in modulating cardiac contractility through its phosphorylation state. Multiple studies have linked MYL2 mutations to hypertrophic cardiomyopathy, making it an important target for cardiovascular research .

What applications are MYL2 antibodies suitable for?

MYL2 antibodies have been validated for numerous research applications including:

• Western Blot (WB)

• Immunoprecipitation (IP)

• Immunohistochemistry (IHC)

• Immunofluorescence (IF)

• Enzyme-Linked Immunosorbent Assay (ELISA)

• Flow Cytometry (FC), including intracellular staining

The versatility of these antibodies makes them valuable tools for investigating MYL2 expression, localization, and modifications across different experimental contexts. When selecting an antibody, researchers should confirm that it has been validated for their specific application of interest.

What are the recommended dilutions for different MYL2 antibody applications?

Optimal antibody dilutions vary depending on the specific application:

It is important to note that these recommendations should serve as starting points. Researchers should perform optimization experiments for their specific samples and experimental conditions . Antibody performance can vary based on tissue type, fixation method, and protein abundance.

How does MYL2 phosphorylation affect cardiac function, and how can this be studied?

MYL2 phosphorylation significantly impacts cardiac muscle contraction mechanics and function. Following phosphorylation by kinases such as MYLK3 and MYLK2, MYL2 increases myosin lever arm stiffness and promotes myosin head diffusion. These molecular changes result in increased maximum contraction force and enhanced calcium sensitivity of contraction .

Interestingly, phosphorylated MYL2 displays a gradient distribution across the heart, with lower phosphorylation in the endocardium and higher phosphorylation in the epicardium. This gradient helps regulate cardiac torsion and workload distribution throughout the cardiac tissue .

To study these phosphorylation patterns, researchers can:

• Use phospho-specific MYL2 antibodies for immunohistochemistry to visualize the spatial distribution

• Perform Western blotting with phospho-specific antibodies alongside total MYL2 antibodies

• Use phosphatase inhibitors during tissue preparation to preserve the phosphorylation state

• Employ cardiac tissue sections from different regions to map the phosphorylation gradient

What protocols optimize Western blot detection of MYL2 in cardiac tissue samples?

For optimal Western blot detection of MYL2 in cardiac tissues, follow these methodological considerations:

• Sample preparation: Extract total tissue protein from fixed samples using specialized kits like the Qproteome FFPE Tissue Kit. Quantify protein concentration using a BCA Protein Assay Kit .

• Gel electrophoresis parameters: Use 12% SDS-PAGE gels to effectively separate MYL2 (approximately 18-20 kDa) .

• Protein loading: Load approximately 15 μg of total protein per lane for adequate MYL2 signal detection .

• Transfer conditions: Transfer proteins to nitrocellulose membranes using standard protocols.

• Blocking: Block membranes with 3-4% milk powder or bovine serum albumin in TBST .

• Antibody incubation: Incubate with primary MYL2 antibody at dilutions between 1:500-1:1000 for approximately 4 hours at room temperature or overnight at 4°C .

• Detection: Use a maximum-sensitivity ECL kit for visualization of the protein bands .

• Controls: Include β-actin (42 kDa) as an internal loading control. MYL2 will appear as a specific band at approximately 18-20 kDa .

• Quantification: Calculate the MYL2/β-actin ratio to normalize expression levels across samples .

How can MYL2 antibodies be used to investigate cardiac disease models?

MYL2 antibodies are valuable tools for investigating cardiac disease models, particularly in hypertrophic cardiomyopathy and heart failure research. Several methodological approaches include:

• Expression analysis in disease models: Western blot analysis using MYL2 antibodies can quantify protein expression changes in disease states. Research has shown that MYL2 levels are significantly reduced in heart failure patients (approximately 69% ± 16% of normal levels), providing a potential biomarker for cardiac dysfunction .

• Tissue localization studies: Immunohistochemistry using MYL2 antibodies can reveal alterations in expression patterns or subcellular localization in diseased tissues.

• Mutation research: Combining MYL2 antibodies with genetically modified models carrying MYL2 mutations can help elucidate the pathophysiological mechanisms of hypertrophic cardiomyopathy.

• Phosphorylation status: Using phospho-specific MYL2 antibodies can reveal changes in the regulatory state of MYL2 in disease conditions, as abnormal phosphorylation patterns may contribute to contractile dysfunction.

What is the optimal immunohistochemistry protocol for MYL2 detection?

For effective immunohistochemical detection of MYL2 in cardiac tissues, follow this optimized protocol:

• Antigen retrieval: Heat tissue sections in TE buffer (pH 9.0) at 95°C for 15 minutes. Alternatively, citrate buffer (pH 6.0) may be used .

• Peroxidase blocking: Treat sections to remove endogenous peroxidases.

• Blocking: Block with 3% bovine serum albumin in PBS at 37°C for 20 minutes .

• Primary antibody: Apply MYL2 antibody at a dilution of 1:400 (or as recommended by the manufacturer) and incubate at 4°C overnight .

• Washing: Wash thoroughly with PBS to remove unbound primary antibody.

• Secondary antibody: Incubate with appropriate species-specific secondary antibody at 37°C for 20 minutes .

• Detection: Visualize antibody binding using the diaminobenzidine (DAB) method.

• Counterstaining: Counterstain nuclei with hematoxylin for approximately 15 seconds .

• Controls: Include negative controls by substituting PBS for primary antibody .

How can I validate the specificity of MYL2 antibodies in my experimental system?

Validating antibody specificity is crucial for generating reliable research data. For MYL2 antibodies, consider these validation approaches:

• Multiple antibody comparison: Use antibodies from different suppliers or those recognizing different epitopes of MYL2.

• Molecular weight verification: Confirm that the detected band in Western blots appears at the expected molecular weight of 18-20 kDa .

• Positive and negative control tissues: Include tissues known to express high levels of MYL2 (heart tissue) and those with minimal expression.

• Peptide competition assay: Pre-incubate the antibody with the immunizing peptide to demonstrate signal specificity.

• Knockdown validation: Use siRNA or CRISPR to reduce MYL2 expression and confirm corresponding reduction in antibody signal.

• Recombinant protein controls: Use purified recombinant MYL2 as a positive control in Western blots.

• Cross-reactivity assessment: Test the antibody against samples from different species if cross-reactivity is claimed by the manufacturer .

What considerations should be made when analyzing MYL2 expression in heart failure models?

When analyzing MYL2 expression in heart failure models, researchers should consider these methodological aspects:

• Sample selection: Carefully select cardiac tissue regions as MYL2 expression and phosphorylation vary across different areas of the heart .

• Normalization strategy: Use appropriate housekeeping proteins (e.g., β-actin) for normalization, but be aware that heart failure may affect expression of some commonly used reference proteins .

• Phosphorylation analysis: Assess both total MYL2 and phosphorylated MYL2 levels, as changes in phosphorylation state may be more significant than total protein changes .

• Quantification methods: Develop consistent quantification approaches. For Western blots, the MYL2/β-actin ratio provides a reliable measure of relative expression .

• Statistical analysis: Heart failure tissues often show increased variability; ensure sufficient sample sizes for statistical power. In published studies, heart failure patients showed MYL2 levels at 69% ± 16% of control group levels (100% ± 21%) .

• Technical replication: Include technical replicates to account for experimental variability, especially important when working with heterogeneous disease samples.

How can I troubleshoot weak or absent MYL2 signal in Western blotting?

When encountering weak or absent MYL2 signal in Western blotting, consider these troubleshooting steps:

• Protein extraction efficiency: Ensure efficient protein extraction, especially from fixed tissue samples. Specialized extraction kits for FFPE tissues may be necessary .

• Protein degradation: Use fresh protease inhibitors in lysis buffers to prevent MYL2 degradation.

• Antibody selection: Different antibody clones target different epitopes; some may be more accessible in denatured samples. Consider antibodies targeting the 91-140 amino acid region or middle region of MYL2 .

• Antibody concentration: Adjust primary antibody concentration. While recommended dilutions range from 1:500 to 1:20000, start with higher concentrations (1:500) when signal is weak .

• Incubation conditions: Extend primary antibody incubation time to overnight at 4°C if room temperature incubation yields weak signals .

• Detection sensitivity: Use maximum-sensitivity ECL kits for enhanced detection of low-abundance proteins .

• Sample loading: Increase the amount of total protein loaded (15-20 μg is recommended) .

• Transfer efficiency: Optimize transfer conditions for low molecular weight proteins (18-20 kDa range) .

What controls should be included when using MYL2 antibodies for immunofluorescence?

For robust immunofluorescence experiments with MYL2 antibodies, incorporate these essential controls:

• Positive tissue controls: Include cardiac tissue sections known to express MYL2 to confirm antibody performance.

• Negative controls: Omit primary antibody but maintain all other steps to identify non-specific secondary antibody binding.

• Isotype controls: Use non-specific antibodies of the same isotype and concentration as the MYL2 antibody to identify potential non-specific binding.

• Absorption controls: Pre-incubate antibody with immunizing peptide before application to tissue sections to confirm specificity.

• Co-localization markers: Include antibodies against known cardiac markers that should or should not co-localize with MYL2 to confirm specific cellular detection.

• Counterstains: Use nuclear counterstains to provide structural context and aid in identifying specific cell types.

• Autofluorescence controls: Examine unstained tissue sections to identify and account for natural tissue autofluorescence, particularly important in cardiac tissues.

How can flow cytometry be optimized for MYL2 detection in cardiac cells?

For optimal flow cytometric detection of MYL2 in cardiac cells, implement these methodological approaches:

• Cell preparation: Carefully dissociate cardiac tissue to obtain single-cell suspensions while preserving cellular integrity.

• Fixation and permeabilization: Since MYL2 is an intracellular protein, effective permeabilization is essential. Use commercially available permeabilization kits specifically optimized for intracellular proteins.

• Antibody concentration: Use approximately 0.40 μg per 10^6 cells in a 100 μl suspension for optimal staining .

• Controls: Include unstained cells, isotype controls, and single-color controls for accurate compensation.

• Viability discrimination: Use viability dyes to exclude dead cells, which can non-specifically bind antibodies.

• Gating strategy: Develop specific gating strategies to identify cardiomyocyte populations based on size, complexity, and cardiac-specific markers.

• Multi-parameter analysis: Combine MYL2 staining with other cardiac markers to identify specific cell populations of interest.

• Validation: Validate flow cytometry results using complementary techniques such as immunofluorescence microscopy.

What are the considerations for quantitative analysis of MYL2 expression levels?

For accurate quantitative analysis of MYL2 expression levels, consider these methodological aspects:

• Normalization approach: Standardize MYL2 expression against stable reference proteins such as β-actin. Calculate the MYL2/β-actin ratio to permit valid comparisons between samples .

• Standard curves: For absolute quantification, develop standard curves using recombinant MYL2 protein.

• Sample handling: Ensure consistent handling of all samples to minimize technical variability in protein extraction and detection.

• Image analysis: For immunohistochemistry or immunofluorescence, use standardized image acquisition settings and validated quantification software.

• Statistical approach: Determine appropriate statistical tests based on data distribution. Published studies have shown that heart failure patients demonstrate MYL2 levels at approximately 69% ± 16% of normal controls .

• Dynamic range: Ensure that detection methods provide adequate dynamic range to capture both high and low expression levels.

• Replication: Include both biological and technical replicates to account for natural and experimental variability.