MYLK2 Antibody

What is MYLK2 and what is its functional significance in muscle physiology?

MYLK2 (Myosin Light Chain Kinase 2) is a calcium/calmodulin-dependent serine/threonine kinase expressed exclusively in adult skeletal and cardiac muscle. The protein has several alternative names including MLCK2, skMLCK, KMLC, and myosin light chain kinase 2 skeletal/cardiac muscle isoform.

MYLK2 plays a crucial role in muscle contractility through the following mechanisms:

• Catalyzes the phosphorylation reaction: ATP + [myosin light-chain] = ADP + [myosin light-chain] phosphate

• Specifically phosphorylates a serine residue in the N-terminus of myosin light chain

• Mediates the excitation-contraction coupling process following calcium release

• Regulates global muscle contraction and cardiac function

This enzyme belongs to the protein kinase superfamily and CAMK Ser/Thr protein kinase family, with a calculated molecular weight of approximately 65 kDa. MYLK2 is implicated in cardiovascular disease processes when dysregulated, making it an important target for both basic muscle physiology research and pathological studies .

What are the optimal applications for MYLK2 antibodies in experimental protocols?

MYLK2 antibodies have been validated across multiple experimental applications with varying degrees of optimization. Based on comprehensive analysis of available products:

When planning experiments, researchers should consider that Western blotting provides the most consistent results across different MYLK2 antibody sources. For applications requiring spatial resolution (IHC/IF), additional validation steps are recommended to confirm specificity .

How should researchers evaluate MYLK2 antibody specificity and performance?

A methodical approach to MYLK2 antibody validation should include:

Positive Control Selection:

Multiple sources confirm successful detection of MYLK2 in:

• Skeletal muscle tissue (human, mouse, rat)

• Jurkat cells

• A549 cells

• MOLT4 whole cell lysate

Negative Control Assessment:

• Use tissues known to lack MYLK2 expression

• Consider knockout/knockdown validation if available

• Include isotype control antibodies in parallel experiments

Critical Validation Parameters:

• Expected molecular weight detection (65 kDa)

• Correct cellular localization (cytoplasmic)

• Cross-reactivity assessment across species

• Batch-to-batch consistency verification

Researchers should perform their own validation experiments rather than relying solely on manufacturer data. This includes titration experiments to determine optimal antibody concentration, which can vary significantly from published recommendations based on specific experimental conditions and detection methods .

What are the key technical considerations for Western blot protocols using MYLK2 antibodies?

Successful Western blot detection of MYLK2 requires specific technical optimizations:

Sample Preparation:

• Use skeletal muscle tissue as positive control

• Optimize protein extraction with buffers containing protease inhibitors

• For cell lines, verify MYLK2 expression levels before experimentation

• Recommended loading: 20-30 μg total protein per lane

Electrophoresis and Transfer Conditions:

• 7.5% SDS-PAGE gels provide optimal separation for MYLK2

• Standard wet transfer protocols are suitable

• Transfer time may need extension due to MYLK2's molecular weight

Detection Protocol:

• Primary antibody incubation: 1:1000-1:2000 dilution range shows consistent results

• Overnight incubation at 4°C improves signal-to-noise ratio

• Secondary antibody selection should match primary host species

• When troubleshooting weak signals, extending primary antibody incubation time is more effective than increasing concentration

The predicted band size for MYLK2 is 65 kDa, though slight variations may occur due to post-translational modifications. Researchers should be aware that some antibodies may detect additional bands, requiring careful interpretation of results .

How does species cross-reactivity influence MYLK2 antibody selection for comparative studies?

MYLK2 sequence homology varies across species, requiring strategic antibody selection for comparative studies:

Cross-Reactivity Profile:

The majority of available MYLK2 antibodies demonstrate reactivity with:

• Human MYLK2 (most commonly validated)

• Mouse MYLK2 (well validated)

• Rat MYLK2 (well validated)

• Limited validation for other species including avian, bovine, and porcine

Epitope Selection Considerations:

Antibodies targeting conserved regions show broader cross-reactivity. Based on available data:

• Antibodies targeting the central region (AA 330-450) show highest cross-species reactivity

• N-terminal epitopes (AA 1-100) generally show more species-specific binding

• C-terminal epitopes provide intermediate cross-reactivity

Methodological Approach for Cross-Species Studies:

• Perform preliminary validation experiments in each species of interest

• Adjust antibody concentration based on species-specific binding efficiency

• Consider epitope sequence alignment analysis before purchasing antibodies

• For novel species applications, select antibodies raised against highly conserved regions

When planning comparative studies across species, researchers should confirm cross-reactivity experimentally rather than relying solely on manufacturer claims. Different applications may require different antibody selections even within the same study .

What methods are available for detecting phosphorylated myosin light chains using MYLK2 antibodies?

Detection of phosphorylated myosin light chains (the substrate of MYLK2) requires specialized approaches:

Phosphorylation Site-Specific Antibodies:

• Anti-phospho myosin light chain antibodies are distinct from MYLK2 antibodies

• These target the specific serine residue phosphorylated by MYLK2

• Require separate validation from MYLK2 antibodies themselves

Kinase Activity Assessment Methods:

• In vitro kinase assays:

  • Immunoprecipitate MYLK2 using anti-MYLK2 antibodies

  • Incubate with purified myosin light chain substrate and ATP

  • Detect phosphorylation via 32P incorporation or phospho-specific antibodies

• Calcium/calmodulin dependence validation:

  • Include EGTA controls to chelate calcium

  • Test activity in presence/absence of calmodulin

  • Establish dose-response relationship with calcium concentration

• Pharmacological inhibition controls:

  • Include MYLK-specific inhibitors (e.g., ML-7, ML-9)

  • Establish dose-dependent inhibition curves

  • Use as negative controls in activity assays

Researchers investigating MYLK2 enzymatic function should consider a complementary approach using both anti-MYLK2 antibodies to detect the enzyme and phospho-specific antibodies to detect its substrate's phosphorylation state .

How can MYLK2 antibodies be optimized for immunohistochemistry and immunofluorescence applications?

Successful detection of MYLK2 in tissue sections requires specific protocol optimizations:

Antigen Retrieval Methods:

• TE buffer (pH 9.0) provides optimal results for most MYLK2 antibodies

• Alternative: citrate buffer (pH 6.0) for antibodies where alkaline retrieval fails

• Heat-induced epitope retrieval (HIER) is generally superior to enzymatic methods

Tissue-Specific Considerations:

• Skeletal muscle: 10% neutral buffered formalin fixation (12-24h) recommended

• Cardiac tissue: shorter fixation times (6-12h) may improve antigen accessibility

• Paraffin sections: 4-6 μm thickness optimal for signal detection

Protocol Optimization Parameters:

• Blocking: 5-10% normal serum from secondary antibody host species

• Primary antibody concentration: Start with 1:100 dilution and titrate as needed

• Incubation time: Overnight at 4°C generally yields superior results

• Secondary detection: Biotin-streptavidin amplification may enhance sensitivity

• Counterstaining: Hematoxylin (blue) provides optimal contrast with DAB detection

For double immunofluorescence studies, researchers should carefully select MYLK2 antibodies raised in different host species than antibodies against other targets of interest to avoid cross-reactivity of secondary antibodies .

What troubleshooting approaches are recommended for inconsistent MYLK2 antibody results?

When facing inconsistent results with MYLK2 antibodies, systematic troubleshooting should address:

Common Western Blot Issues:

• No signal detected:

  • Verify MYLK2 expression in sample (use positive control tissue)

  • Test primary antibody viability with dot blot

  • Increase protein loading (up to 50 μg)

  • Extend exposure time or switch to more sensitive detection method

• Multiple bands:

  • Optimize blocking conditions (increase BSA/milk percentage)

  • Reduce primary antibody concentration

  • Verify antibody specificity with blocking peptide

  • Consider whether bands represent isoforms or degradation products

Immunohistochemistry Troubleshooting:

• High background:

  • Increase blocking time/concentration

  • Reduce primary antibody concentration

  • Include 0.1-0.3% Triton X-100 in wash buffers

  • Extend wash steps (minimum 3x15 minutes)

• Weak or absent signal:

  • Test alternative antigen retrieval methods

  • Increase primary antibody concentration

  • Extend incubation time to overnight at 4°C

  • Consider signal amplification systems

Storage and Handling Considerations:

• Avoid repeated freeze-thaw cycles (aliquot upon receipt)

• For long-term storage, maintain at -20°C

• For frequent use, store at 4°C for up to one month

• Centrifuge antibody briefly before opening to collect solution

Detailed record-keeping of experimental conditions facilitates systematic troubleshooting and allows for identification of variables affecting antibody performance.

How can MYLK2 antibodies be utilized for investigating cardiac pathophysiology?

MYLK2 antibodies provide valuable tools for examining cardiac disease mechanisms:

Hypertrophic Cardiomyopathy (HCM) Research Applications:

• MYLK2 mutations have been associated with HCM (identified as CMH10)

• Antibodies can detect altered expression/localization in disease models

• Quantitative analysis of MYLK2 levels correlates with disease progression

Methodology for Cardiac Tissue Analysis:

• Tissue preparation:

  • Fresh-frozen cardiac tissue provides superior antigen preservation

  • Consider specific fixation protocols for MYLK2 detection

  • Sectioning thickness: 5-8 μm optimal for cardiac tissue

• Co-localization studies:

  • Combine MYLK2 antibodies with markers of:

    • Sarcomeric structures (α-actinin, troponin)

    • Calcium handling proteins (SERCA2a, RyR2)

    • Pathological remodeling (fibrosis markers)

• Quantitative analysis approaches:

  • Western blot for total MYLK2 expression changes

  • IHC/IF with digital image analysis for spatial distribution alterations

  • Correlation with functional parameters (contractility measurements)

Researchers should consider that MYLK2 expression and activity may be differentially regulated in cardiac versus skeletal muscle, necessitating tissue-specific protocol optimizations and controls .

What advanced research applications utilize MYLK2 antibodies beyond conventional detection methods?

Beyond standard detection applications, MYLK2 antibodies enable sophisticated research approaches:

Proximity Ligation Assay (PLA):

• Allows visualization of protein-protein interactions in situ

• Can detect MYLK2 interactions with calcium/calmodulin and its substrates

• Requires careful selection of antibody pairs raised in different host species

ChIP-Sequencing Applications:

• ChIP-grade MYLK2 antibodies can identify transcriptional regulatory mechanisms

• Enables genome-wide binding site identification

• Requires validation for chromatin immunoprecipitation specificity

Single-Cell Analysis:

• Flow cytometry applications (optimized fixation/permeabilization required)

• Mass cytometry (CyTOF) for multi-parameter analysis

• Single-cell Western blotting for heterogeneity assessment

Live-Cell Imaging Approaches:

• Use of fluorescently conjugated MYLK2 antibody fragments

• Requires validation of antibody effect on MYLK2 function

• Alternative: correlation with genetically encoded MYLK2 fusion proteins

These advanced applications generally require extensive validation and optimization beyond standard protocols. Researchers should conduct preliminary experiments to verify antibody suitability for these specialized techniques before proceeding with full-scale studies .

How does antibody format selection impact experimental outcomes for MYLK2 detection?

The format of MYLK2 antibodies significantly influences experimental results:

Unconjugated vs. Conjugated Formats:

• Unconjugated: Most versatile, allows custom secondary detection

• Direct conjugates (e.g., biotin, fluorophores): Reduce steps, enable multiplexing

• Enzyme conjugates (HRP, AP): Provide direct detection without secondaries

Format Selection Considerations by Application:

Methodological Impact of Format Selection:

• For multiplex fluorescence, select formats with non-overlapping emission spectra

• For chromogenic IHC, enzyme conjugates or biotin systems provide optimal results

• Consider background concerns when using direct conjugates in tissues with high autofluorescence

The biotin-conjugated MYLK2 antibody format offers particular advantages for techniques requiring signal amplification through streptavidin-based detection systems, while fluorophore conjugates are optimal for co-localization studies .

What are the key methodological considerations when studying MYLK2 in different muscle types?

MYLK2 expression and function varies between muscle types, requiring tailored experimental approaches:

Tissue-Specific Expression Patterns:

• Skeletal muscle: Highest expression levels (fast-twitch > slow-twitch fibers)

• Cardiac muscle: Significant expression but lower than skeletal muscle

• Smooth muscle: Minimal MYLK2 (predominantly expresses MYLK1)

• Non-muscle tissues: Negligible MYLK2 expression

Methodological Approaches for Comparative Studies:

• Fiber-type specific analysis:

  • Co-staining with fiber-type markers (MHC isoforms)

  • Serial section analysis for fiber typing

  • Correlation of MYLK2 levels with contractile properties

• Tissue preparation considerations:

  • Skeletal muscle: Critical to maintain fiber orientation during sectioning

  • Cardiac muscle: Attention to regional differences (atria vs. ventricles)

  • Control for post-mortem degradation effects on phosphorylation status

• Functional correlation approaches:

  • Combine antibody detection with muscle physiology measurements

  • Ex vivo muscle contractility studies

  • In vivo analysis in genetic models (knockout/knockin)