MYLPF Antibody

What is MYLPF and what is its function in muscle tissue?

MYLPF (myosin light chain, phosphorylatable, fast skeletal muscle) is a critical regulatory component of the myosin complex in skeletal muscle. It functions as a phosphorylatable light chain within the myosin hexamer, which consists of two heavy chain subunits, two non-phosphorylatable light chain subunits, and two phosphorylatable light chain subunits . MYLPF plays an essential role in muscle contraction by regulating the interaction between myosin and actin filaments through calcium/calmodulin-dependent phosphorylation .

Research has demonstrated that MYLPF is crucial for myofibril assembly in fast-twitch muscle fibers. The protein dosage of MYLPF directly correlates with myofibril width and proper assembly . In zebrafish models, complete absence of MYLPF (through double knockout of mylpfa and mylpfb genes) results in total failure of myofibril formation, demonstrating its indispensable role in muscle development . The protein interacts with myosin heavy chains and actin to generate the force required for cellular movements, making it central to muscle function and contractility .

What applications are MYLPF antibodies validated for in muscle research?

MYLPF antibodies have been validated for multiple research applications, with specific optimization parameters for each technique:

For immunohistochemistry applications, antigen retrieval techniques significantly impact results. TE buffer at pH 9.0 is suggested for optimal antigen retrieval, with citrate buffer at pH 6.0 serving as an alternative option . When working with any of these applications, researchers should note that dilution optimization is often sample-dependent, requiring titration in each experimental system for optimal results .

Western blot detection typically reveals a single band at approximately 19-20 kDa, corresponding to the MYLPF protein . For specialized applications like zebrafish research, antibodies can distinguish between the Mylpfa (16 kD) and Mylpfb (18 kD) isoforms when run on appropriate gradient gels .

How does MYLPF expression correlate with muscle fiber types and development?

MYLPF demonstrates a highly specific expression pattern that correlates with muscle fiber types and developmental stages:

Muscle Fiber Type Specificity

MYLPF is exclusively expressed in fast-twitch muscle fibers, with no expression in slow-twitch muscle . This fiber-type specificity makes MYLPF antibodies valuable tools for distinguishing fast-twitch from slow-twitch muscle in research applications. Immunohistochemistry analysis consistently shows high MYLPF expression in skeletal muscle tissue (containing fast-twitch fibers) and minimal expression in tissues dominated by smooth muscle, such as the duodenum .

Developmental Expression Patterns

The expression of MYLPF begins early in development and follows a regulated pattern:

• In zebrafish, expression of mylpfa and mylpfb is detectable as early as 20 hours post fertilization (hpf) in medial fast-twitch fibers

• At 24 hpf, there is a 6:1 expression ratio between mylpfa and mylpfb transcripts

• This ratio decreases to 3:1 by 36 hpf, primarily due to increasing mylpfb expression

• Protein abundance directly correlates with transcript levels throughout development

• In humans, MYLPF expression is detected in both fetal and adult skeletal muscle

This developmental regulation is functionally significant, as early MYLPF expression levels predict the extent of myofibril formation in later developmental stages . The dosage-dependent effects of MYLPF on myofibril width are maintained throughout embryonic development, highlighting its critical role in muscle formation and maturation.

What are the species reactivity profiles for commonly used MYLPF antibodies?

MYLPF antibodies demonstrate varying species reactivity profiles, with most antibodies showing cross-reactivity across multiple mammalian species due to the high conservation of the MYLPF protein sequence:

When selecting an antibody for cross-species applications, researchers should consider both validated reactivity (experimentally confirmed) and predicted reactivity (based on sequence homology). For novel applications in untested species, validation experiments should be performed to confirm specificity and appropriate working conditions.

The broad cross-species reactivity of these antibodies makes them valuable tools for comparative studies across different model organisms, though optimization may be required when transitioning between species.

What is the relationship between MYLPF dosage and myofibril formation?

Research has established a direct, proportional relationship between MYLPF protein dosage and myofibril formation in fast-twitch muscle:

Dose-Dependent Effects on Myofibril Width

Studies in zebrafish demonstrate that the combined dosage of Mylpf proteins (from mylpfa and mylpfb genes) determines myofibril width in a linear relationship . Complete absence of both Mylpf genes results in total failure of myofibril assembly, while partial reduction (as in heterozygous mutants) leads to proportionally reduced myofibril width .

Quantitative Relationship

The relationship between MYLPF dosage and myofibril formation follows a clear pattern:

• 0% dose (double knockout): Complete absence of myofibrils

• 25% dose (homozygous/heterozygous combination): Severe myofibril defects

• 50% dose (double heterozygote): Moderately reduced myofibril width

• 100% dose (wild-type): Normal myofibril development

Rescue Experiments

Transgenic expression of MYLPF-GFP in mylpfa mutants restores myofibril width in direct proportion to the expression level (measured by GFP fluorescence intensity) . This linear relationship provides strong evidence for dose-dependent function.

Pathological Variants

MYLPF variants associated with Distal Arthrogryposis (DA) invert the dose-relationship, with increasing levels of mutant protein causing progressive reduction in myofibril width . The G163S variant (which affects MyHC interaction) demonstrates antimorphic activity, while the C157F variant shows reduced function with mild antimorphic effects .

This dosage sensitivity makes MYLPF levels a critical determinant of muscle development and function, with significant implications for understanding both normal physiology and pathological conditions.

How can researchers validate MYLPF antibody specificity?

Validating antibody specificity is crucial for reliable research results. For MYLPF antibodies, several validation strategies have proven effective:

Genetic Model Validation

One of the most rigorous approaches utilizes genetic models with known mutations or knockouts of MYLPF:

• Zebrafish mylpfa mutants show absence of the Mylpfa band (16 kD) on Western blots while retaining the Mylpfb band (18 kD), confirming antibody specificity

• Proper genetic validation requires:

  • Sorting animals by behavioral phenotypes (as MYLPF mutations affect movement)

  • Confirming genotypes through PCR or other genetic validation methods

  • Comparing protein expression patterns between wild-type and mutant samples

Multiple Antibody Validation

Using multiple antibodies targeting different epitopes of MYLPF provides complementary validation:

• Primary validation antibody targets MYLPF

• Secondary validation antibodies target known interaction partners (e.g., MyHC)

• Comparing results across antibodies confirms specific detection

Technical Considerations for Western Blot Validation

For Western blot validation, several technical factors ensure reliable results:

• Sample preparation: Homogenize tissues using appropriate buffers (e.g., Bolt solution) and mechanical disruption (0.5 mm zirconium beads)

• Gel selection: Use gradient gels (4-20%) or "any kD" precast polyacrylamide gels that can resolve closely related proteins

• Loading controls: Include appropriate loading controls and reference proteins (e.g., MyHC) for normalization

• Detection systems: Use dual-color detection systems (e.g., Odyssey Infrared Imager) for simultaneous visualization of target and control proteins

Orthogonal Validation

Some MYLPF antibodies, such as the Novus Biologicals Fast skeletal myosin light chain 2 Antibody (NBP2-62625), have undergone orthogonal validation, correlating protein detection with RNA-seq data from the same tissues . This approach confirms that antibody staining patterns match gene expression profiles.

Thorough validation using these approaches ensures that experimental results truly reflect MYLPF biology rather than non-specific interactions or artifacts.

How do mutations in MYLPF affect myofibril assembly and muscle function?

Mutations in MYLPF have profound and varied effects on myofibril assembly and muscle function, revealing the protein's critical role in muscle development:

Complete Loss of Function

In zebrafish double mutants (mylpfa-/-; mylpfb-/-), complete loss of MYLPF function results in:

• Total failure of myofibril formation in fast-twitch muscle

• Only scattered thick filaments and I-Z-I bodies visible by transmission electron microscopy

• Absence of organized sarcomeric structures

Partial Loss of Function

Single gene mutations produce varied phenotypes:

• mylpfa-/- mutants show severely reduced fast-twitch myofibrils and lose high-speed movement capabilities

• mylpfb-/- mutants display normal myofibrils, indicating functional redundancy between the genes

• mylpfa+/-; mylpfb+/- double heterozygotes exhibit moderately reduced myofibril width, demonstrating dosage sensitivity

Functional Adaptation

Despite severe structural defects, mylpfa mutant zebrafish can still perform persistent slow movement, allowing them to cover similar distances over time as wild-type siblings. This adaptation suggests compensatory mechanisms through slow-twitch muscle function .

Human Disease Mutations

MYLPF mutations associated with Distal Arthrogryposis (DA) demonstrate distinct mechanisms:

• G163S mutation (affecting MyHC interaction): Cannot rescue myofibril formation and shows antimorphic effects

• C157F mutation (affecting protein structure): Shows reduced function with mild antimorphic effects

Molecular Mechanisms

MYLPF mutations impact muscle development through multiple mechanisms:

These findings highlight how MYLPF mutations can cause structural defects in muscle that manifest as functional impairments in movement and contractility, with implications for both developmental disorders and potential therapeutic approaches.

What are the optimal sample preparation methods for MYLPF detection?

Optimal sample preparation methods for MYLPF detection vary by application and tissue type, with specific considerations required for each technique:

Western Blot Sample Preparation

For effective Western blot detection of MYLPF:

• Tissue collection:

  • For developmental studies, collect pools of 10-40 animals depending on stage

  • For muscle-specific studies, isolate skeletal muscle tissue where MYLPF is highly expressed

• Homogenization:

  • Use mechanical disruption (0.5 mm zirconium beads) in appropriate buffer (e.g., Bolt solution)

  • Include protease inhibitors to prevent degradation

• Sample processing:

  • Run samples on 4-20% gradient acrylamide gels or "any kD" precast gels to resolve MYLPF (~19 kDa)

  • Transfer to PVDF membranes for optimal protein binding

  • Block with 5% nonfat dry milk overnight at 4°C

Immunohistochemistry Sample Preparation

For IHC applications with MYLPF antibodies:

• Tissue fixation:

  • Formalin fixation and paraffin embedding preserve tissue architecture

  • Frozen sections may provide better antigen preservation for some applications

• Antigen retrieval:

  • TE buffer at pH 9.0 is recommended as the primary retrieval method

  • Citrate buffer at pH 6.0 serves as an alternative option

  • For the NBP2-62625 antibody, HIER pH 6 retrieval is specifically recommended

• Antibody dilution:

  • For standard IHC: 1:20-1:200 dilution range

  • For IHC-Paraffin: 1:500-1:1000 dilution range

  • Optimize dilution for each tissue type and detection system

Tissue Selection Considerations

Due to MYLPF's specific expression pattern:

• For positive control tissues, use skeletal muscle samples rich in fast-twitch fibers

• For negative control or comparison tissues, consider slow-twitch-dominated muscles or non-muscle tissues like duodenum

• When studying developmental processes, carefully stage samples as MYLPF expression changes throughout development

These optimized preparation methods ensure reliable detection of MYLPF across different experimental contexts, facilitating accurate analysis of its expression, localization, and function.

How can MYLPF antibodies be used to study myopathies and muscle disorders?

MYLPF antibodies offer valuable tools for investigating myopathies and muscle disorders through multiple research approaches:

Distal Arthrogryposis Research

MYLPF mutations are implicated in Distal Arthrogryposis (DA), a congenital contracture syndrome:

• Mutation analysis:

  • Antibodies can detect expression levels and localization of DA-associated MYLPF variants

  • Research has characterized functional impacts of variants including G163S (dominant, severely disruptive) and C157F (recessive, partially functional)

• Functional studies:

  • Transgenic expression of human MYLPF variants in zebrafish models allows functional assessment

  • MYLPF antibodies confirm expression levels and correlate them with phenotypic effects

Muscle Development Disorders

For investigating developmental muscle disorders:

• Dosage analysis:

  • MYLPF antibodies can quantify protein levels in patient samples

  • Reduced MYLPF levels correlate with myofibril width defects and functional impairment

• Structural assessment:

  • Immunofluorescence with MYLPF antibodies visualizes sarcomere organization

  • Colocalization with other sarcomeric markers helps characterize structural defects

Model System Applications

In animal and cellular models:

• Zebrafish models:

  • MYLPF antibodies distinguish between Mylpfa (16 kD) and Mylpfb (18 kD)

  • Western blot analysis quantifies isoform-specific expression during development and in disease models

• Muscle cell cultures:

  • Immunocytochemistry with MYLPF antibodies tracks myofibril assembly in differentiating muscle cells

  • Expression patterns correlate with functional maturation of the contractile apparatus

Therapeutic Development

For evaluating potential therapeutic approaches:

• Rescue experiments:

  • MYLPF antibodies assess protein expression following genetic interventions

  • Restoration of MYLPF levels can be correlated with functional recovery

• Drug screening:

  • Compounds affecting MYLPF expression or function can be evaluated using antibody-based detection

  • Changes in MYLPF levels or localization may serve as biomarkers for treatment efficacy

By providing specific detection of MYLPF in various experimental systems, these antibodies facilitate research into the molecular mechanisms of muscle disorders and the development of potential therapeutic strategies.

What are the best methods for quantifying MYLPF expression in comparative studies?

Multiple methodological approaches enable precise quantification of MYLPF expression in comparative research studies:

Western Blot Quantification

For quantitative Western blot analysis of MYLPF:

• Detection systems:

  • Infrared fluorescence imaging (e.g., Odyssey Infrared Imager) provides superior quantitative performance compared to chemiluminescence

  • Dual-color detection allows simultaneous visualization of MYLPF and control proteins

• Software analysis:

  • GelBox software and FIJI/ImageJ provide accurate band intensity quantification

  • Apply background subtraction and normalization to reference proteins

• Technical considerations:

  • Use appropriate antibody dilutions (1:500-1:2000)

  • Include gradient standards for calibration when absolute quantification is needed

  • Run biological replicates to account for sample variation

Immunohistochemistry/Immunofluorescence Quantification

For tissue-level quantification:

• Image acquisition:

  • Use consistent exposure settings across all comparative samples

  • Acquire multiple fields per sample to account for regional variation

• Analysis approaches:

  • Mean fluorescence intensity measurement in defined regions of interest

  • Fiber-by-fiber analysis for heterogeneous tissues

  • Colocalization analysis with other sarcomeric markers

• Validation:

  • Compare IHC results with corresponding RNA-seq or qPCR data

  • Include known positive and negative control tissues in each experiment

In Vivo Fluorescence Correlation

For dynamic studies in live systems:

• Fluorescent fusion proteins:

  • GFP-tagged MYLPF enables in vivo quantification

  • Fluorescence intensity directly correlates with functional effects on myofibril width

• Application in mutant backgrounds:

  • Expression in mylpfa-/- mutants provides clean background for quantification

  • Linear correlation between GFP signal intensity and myofibril rescue extent

Normalization Strategies

For reliable comparative analysis:

• Internal controls:

  • Normalize MYLPF levels to stable reference proteins (e.g., MyHC)

  • Use multiple reference proteins for robust normalization

• Sample considerations:

  • Account for developmental stage-specific expression patterns

  • Consider fiber-type composition when comparing different muscle groups

These methodological approaches provide researchers with multiple options for quantifying MYLPF protein levels across different experimental contexts, enabling reliable comparative studies of expression patterns in development, disease, and potential therapeutic interventions.