MYF5 Antibody, HRP conjugated

What is MYF5 and what biological functions does it serve?

MYF5 (Myogenic Factor 5) functions as a transcriptional activator that promotes transcription of muscle-specific target genes and plays a critical role in muscle differentiation. It co-occupies muscle-specific gene promoter core regions during myogenesis alongside MYOG and MYOD1. Importantly, MYF5 helps maintain the balance between myoblast proliferation and differentiation, ensuring effective muscle regeneration and repair. Dysfunctional MYF5 can contribute to disease progression in muscle-related pathologies . The protein induces fibroblasts to differentiate into myoblasts and is characterized as a sequence-specific DNA-binding protein .

What are the structural and biochemical properties of human MYF5 protein?

Human MYF5 protein consists of 255 amino acid residues with a molecular mass of approximately 28.3 kDa. It belongs to the basic helix-loop-helix (bHLH) family of transcription factors (specifically class C, bHLHc2) and is primarily localized in the nucleus where it performs its transcriptional regulatory functions . The protein contains DNA-binding domains that enable its interaction with specific promoter regions of muscle-related genes, facilitating its role in myogenesis and muscle differentiation .

What is the advantage of using an HRP-conjugated MYF5 antibody compared to unconjugated versions?

HRP (Horseradish Peroxidase)-conjugated MYF5 antibodies offer direct detection capabilities without requiring secondary antibody incubation, which streamlines experimental workflows and reduces background noise. This direct detection system is particularly advantageous for ELISA applications where the conjugated enzyme can immediately react with substrate to produce a measurable signal . This approach reduces experiment time, eliminates potential cross-reactivity issues from secondary antibodies, and can enhance sensitivity in assays where detection amplification is not required.

What are the primary applications for MYF5 antibody, HRP conjugated?

MYF5 antibody with HRP conjugation is primarily optimized for ELISA applications, allowing for direct detection of the target protein . While unconjugated MYF5 antibodies are suitable for multiple applications including Western Blot (WB), Immunocytochemistry (ICC), Immunofluorescence (IF), and Immunohistochemistry (IHC), the HRP-conjugated version is specifically designed for enhanced performance in enzyme-linked immunosorbent assays where direct enzymatic detection is advantageous . The conjugated antibody eliminates the need for secondary antibody incubation steps, streamlining the experimental workflow.

How should sample preparation be optimized for detecting MYF5 in cellular contexts?

For optimal detection of MYF5 in cellular contexts, fixation with paraformaldehyde followed by permeabilization with saponin is recommended, as demonstrated in flow cytometry protocols . For immunocytochemistry applications, immersion fixation of cells (such as C2C12 mouse myoblast cell line) followed by antibody incubation at 10 μg/mL for approximately 3 hours at room temperature has been shown to be effective . Nuclear counterstaining with DAPI is advisable since MYF5 is primarily localized in the nucleus. When using HRP-conjugated antibodies for ELISA, careful blocking steps are essential to minimize background signal.

What is the recommended protocol for ELISA using MYF5 antibody, HRP conjugated?

For ELISA using MYF5 antibody with HRP conjugation, the following methodology is recommended:

• Coat microplate wells with capture antigen or antibody (depending on sandwich or direct ELISA format)

• Block non-specific binding sites with appropriate blocking buffer (typically containing BSA or non-fat milk)

• Add samples containing MYF5 protein

• Add HRP-conjugated MYF5 antibody at experimentally optimized dilution (typically starting with manufacturer's recommendation)

• Incubate at the specified temperature and duration (often 1-2 hours at room temperature or overnight at 4°C)

• Wash thoroughly to remove unbound antibody

• Add HRP substrate (TMB, ABTS, or OPD)

• Measure colorimetric signal using a spectrophotometer at appropriate wavelength

Optimal antibody concentration should be determined experimentally for each specific application and sample type.

How can non-specific binding be minimized when using MYF5 antibody, HRP conjugated?

To minimize non-specific binding when using HRP-conjugated MYF5 antibodies:

• Optimize blocking conditions using 3-5% BSA or non-fat dry milk in PBST/TBST

• Ensure thorough washing between steps (at least 3-5 washes)

• Pre-absorb the antibody with irrelevant proteins if cross-reactivity is observed

• Include appropriate negative controls (samples lacking MYF5 expression)

• Titrate antibody concentration to determine optimal signal-to-noise ratio

• Consider adding 0.1-0.3% Tween-20 in washing buffers to reduce hydrophobic interactions

• For ELISA applications, evaluate different plate types (high binding vs. medium binding)

The purity of sample preparation significantly impacts specificity, so proper sample processing protocols should be followed carefully.

What are common issues when detecting MYF5 in muscle tissue samples and how can they be resolved?

Common issues when detecting MYF5 in muscle tissue samples include:

• Background signal: Optimize blocking conditions using 3-5% BSA or non-fat milk and increase washing steps

• Weak signal: Consider antigen retrieval methods for fixed tissues; heat-induced epitope retrieval in citrate buffer (pH 6.0) is often effective

• Variable expression levels: MYF5 expression varies with muscle developmental stage and regeneration status; appropriate positive controls representing similar physiological conditions should be included

• Cross-reactivity with other MRFs: Due to structural similarities with other myogenic regulatory factors, validate antibody specificity using known positive and negative controls

• Nuclear localization challenges: Ensure proper permeabilization methods are used, as MYF5 is primarily nuclear; saponin permeabilization has shown good results

For HRP-conjugated antibodies specifically, substrate selection and development time should be carefully optimized to maintain signal-to-noise ratio.

How should dilution optimization be conducted for MYF5 antibody, HRP conjugated?

Dilution optimization for HRP-conjugated MYF5 antibody should follow a systematic titration approach:

• Prepare a dilution series (typically 1:500, 1:1000, 1:2000, 1:5000, 1:10000) of the antibody

• Run parallel assays with identical positive control samples containing known MYF5 expression

• Include negative controls lacking MYF5 expression for each dilution

• Calculate signal-to-noise ratio for each dilution (positive signal divided by background signal)

• Select the dilution that provides the highest signal-to-noise ratio while maintaining sufficient absolute signal intensity

• Validate the selected dilution across multiple experimental replicates

• Consider that optimal dilution may vary between different lots of antibody or different sample types

As stated in resource , "Optimal dilutions should be determined by each laboratory for each application," emphasizing the importance of empirical optimization.

How can Western blot results using MYF5 antibodies be accurately interpreted?

For accurate interpretation of Western blot results using MYF5 antibodies:

• Expected molecular weight: Human MYF5 protein has a theoretical molecular weight of 28.3 kDa , but post-translational modifications may cause slight variations

• Band pattern analysis: Single band at expected molecular weight indicates specificity; multiple bands may indicate isoforms, degradation products, or non-specific binding

• Tissue specificity: Strongest expression is typically observed in skeletal muscle tissue, particularly during development or regeneration

• Developmental timing: Expression levels vary significantly during different developmental stages; fetal skeletal muscle typically shows higher expression than adult tissue except during regeneration

• Quantification methodology: For semi-quantitative analysis, normalize MYF5 signal to housekeeping proteins (β-actin, GAPDH) and use digital image analysis software

For HRP-conjugated antibodies specifically used in other applications like ELISA, similar considerations for specificity and expression patterns apply when interpreting results.

What controls should be included when working with MYF5 antibody, HRP conjugated?

When working with MYF5 antibody, HRP conjugated, the following controls should be included:

• Positive tissue control: C2C12 mouse myoblast cell line or skeletal muscle tissue (particularly fetal tissue) which has demonstrated MYF5 expression

• Negative tissue control: Cell types or tissues not expressing MYF5 (e.g., fully differentiated tissues not undergoing regeneration)

• Antibody controls:

  • Primary antibody omission control

  • Isotype control antibody at equivalent concentration

  • Blocking peptide competition assay to confirm specificity

• Signal development control: Include a known positive sample developed for different time intervals when using HRP substrate to optimize signal-to-background ratio

• Cross-reactivity control: If working with multiple species, include samples from various species to confirm cross-reactivity as claimed by manufacturer

These controls are essential for validating experimental results and troubleshooting potential issues with antibody specificity or protocol optimization.

How can MYF5 expression be analyzed in the context of muscle regeneration studies?

In muscle regeneration studies, MYF5 expression analysis should be approached as follows:

• Temporal profiling: MYF5 expression increases in satellite cells during early regeneration phases; establish a time-course analysis (e.g., 0, 1, 3, 5, 7, 14 days post-injury)

• Co-localization analysis: Perform dual immunostaining of MYF5 with satellite cell markers (Pax7) and other myogenic factors (MyoD, myogenin) to track progression through the regeneration program

• Quantitative assessment:

  • Count MYF5+ cells per field or per fiber

  • Measure fluorescence intensity in immunofluorescence studies

  • Quantify protein levels by Western blot or ELISA normalized to total protein

• Spatial distribution: Analyze MYF5 expression relative to injury site and regenerating regions

• Correlation with functional recovery: Link MYF5 expression patterns with physiological or histological markers of muscle regeneration

MYF5 expression helps maintain the balance between myoblast proliferation and differentiation, making it a crucial marker for monitoring regeneration efficiency.

How can MYF5 antibody, HRP conjugated be used to investigate the interplay between MYF5 and other myogenic regulatory factors?

The HRP-conjugated MYF5 antibody can be employed in multi-faceted approaches to investigate interplay with other myogenic regulatory factors:

• Sequential ChIP assays: Use ELISA-based detection to analyze the co-occupancy of MYF5 with MYOD1 and MYOG on muscle-specific gene promoters

• Protein complex detection: Develop sandwich ELISA systems using HRP-conjugated MYF5 antibody paired with antibodies against interacting partners

• Temporal expression analysis: Utilize the antibody in time-course studies to establish expression sequence of MRFs during myogenesis or regeneration

• Comparative quantification: Develop multiplexed ELISA approaches to simultaneously quantify different MRFs using differentially conjugated antibodies

• Signal pathway investigation: Combine with phospho-specific antibodies to correlate MYF5 expression with activation of relevant signaling pathways

The high specificity of properly validated HRP-conjugated antibodies makes them valuable tools for investigating these complex molecular interactions in myogenesis.

What methodological considerations are important when designing experiments to study MYF5's role in satellite cell activation?

When designing experiments to study MYF5's role in satellite cell activation:

• Isolation timing: Satellite cells should be isolated at specific time points following injury or activation stimulus to capture the dynamic expression of MYF5

• Quiescence preservation: Minimize activation during isolation by using gentle dissociation methods and maintaining cold conditions

• Purification methodology: FACS sorting for satellite cell markers (Pax7+/Myf5-/+) with careful gating strategies

• Culture conditions: Use suspension cultures or specialized matrices that preserve satellite cell characteristics rather than triggering immediate differentiation

• Single-cell analysis: Consider coupling antibody detection with single-cell approaches to address heterogeneity in satellite cell populations

• In vivo tracking: For animal studies, consider EdU labeling combined with MYF5 detection to track proliferation of activated satellite cells

• Detection sensitivity: Optimize detection protocols for low abundance proteins in quiescent vs. activated states

These methodological considerations help capture the true biological role of MYF5 in satellite cell biology while minimizing experimental artifacts.

How can chromatin immunoprecipitation be adapted using MYF5 antibody to identify novel target genes?

While HRP-conjugated antibodies are not typically used for ChIP, the following adapted methodology could be employed using unconjugated MYF5 antibodies with subsequent detection systems:

• Crosslinking optimization: For transcription factors like MYF5, dual crosslinking with DSG (disuccinimidyl glutarate) followed by formaldehyde can improve capture of protein-DNA complexes

• Sonication parameters: Optimize sonication conditions to generate DNA fragments of 200-500bp, ideal for resolving binding sites

• Antibody validation: Validate MYF5 antibody specificity for ChIP applications using positive controls (known MYF5 binding sites)

• Sequential ChIP (Re-ChIP): Perform sequential immunoprecipitation with antibodies against MYF5 followed by other myogenic factors to identify co-regulated targets

• ChIP-seq analysis: Couple with next-generation sequencing to identify genome-wide binding sites

• Motif analysis: Analyze recovered sequences for consensus binding motifs using bioinformatic tools

• Validation studies: Confirm novel targets using reporter assays with wild-type and mutated binding sites

This approach enables comprehensive identification of the MYF5 targetome in different cellular contexts, advancing understanding of its regulatory networks.

How can MYF5 antibody be used to evaluate muscle disorders and their progression?

MYF5 antibody can be utilized in several approaches to evaluate muscle disorders:

• Diagnostic biomarker assessment: Quantify MYF5 expression in muscle biopsies from patients with various myopathies compared to healthy controls

• Regenerative capacity evaluation: Assess MYF5+ satellite cell populations in dystrophic muscles to determine regenerative potential

• Therapy response monitoring: Track changes in MYF5 expression following therapeutic interventions to assess impact on myogenic programming

• Disease progression markers: Correlate MYF5 expression patterns with clinical parameters and disease progression

• Comparative analysis: Develop immunohistochemistry scoring systems to standardize MYF5 evaluation across patient cohorts

Dysfunctional MYF5 can contribute to disease progression in muscle-related pathologies, making its detection valuable for understanding pathophysiological mechanisms.

What methodological approaches allow for distinguishing between active and reserve satellite cell populations using MYF5 detection?

To distinguish between active and reserve satellite cell populations:

• Co-immunostaining protocol: Combine MYF5 antibody detection with Pax7 (present in all satellite cells) and MyoD (active cells) to identify:

  • Pax7+/MYF5-/MyoD- (quiescent reserve cells)

  • Pax7+/MYF5+/MyoD- (early activated cells)

  • Pax7+/MYF5+/MyoD+ (committed actively proliferating cells)

• EdU pulse-chase labeling: Combine with MYF5 detection to identify proliferating vs. non-proliferating MYF5+ populations

• Flow cytometry panel design: Develop multi-parameter flow cytometry to quantify these populations in cell suspensions

• Single-cell sequencing integration: Validate antibody-based classifications with transcriptomic profiles

• Lineage tracing validation: In animal models, combine with genetic lineage tracing of Myf5-expressing cells

This multi-parameter approach allows for functional classification of satellite cell subpopulations and their dynamics during homeostasis and regeneration.

How should researchers address conflicting data when MYF5 protein detection does not correlate with mRNA expression levels?

When facing discrepancies between MYF5 protein and mRNA levels:

• Technical validation:

  • Confirm antibody specificity using knockout/knockdown controls

  • Validate primer specificity for mRNA detection

  • Use multiple antibodies targeting different epitopes

• Biological explanations exploration:

  • Investigate post-transcriptional regulation (miRNAs targeting MYF5 mRNA)

  • Examine protein stability and degradation pathways

  • Assess translation efficiency using polysome profiling

• Temporal dynamics consideration:

  • Perform high-resolution time-course studies as protein may lag behind mRNA

  • Consider pulsed expression followed by protein persistence

• Methodological approaches:

  • Employ absolute quantification methods for both protein and mRNA

  • Perform single-cell analyses to address population heterogeneity

  • Use reporter systems to track real-time expression dynamics

These discrepancies often reveal important regulatory mechanisms controlling MYF5 expression and function during myogenesis.