MYF5 Antibody, FITC conjugated

Basic Research Questions

• What is MYF5 and what role does it play in muscle development?

MYF5 (Myogenic factor 5) is a transcription factor belonging to the myogenic regulatory factor (MRF) family, characterized by a basic helix-loop-helix (bHLH) domain. It functions as a transcriptional activator that promotes the expression of muscle-specific genes . MYF5 is expressed first among MRFs during early developmental stages and plays a crucial role in determining myoblast specificity and regulating cell proliferation . It works cooperatively with other myogenic factors including MYOD1 and MYOG to co-occupy muscle-specific gene promoter regions during myogenesis . MYF5 has the remarkable ability to induce non-muscle cells such as fibroblasts to differentiate into myoblasts, demonstrating its potent myogenic regulatory activity . In adult muscle, different levels of MYF5 expression (MYF5High and MYF5Low) mark distinct subpopulations of muscle stem cells with varying regenerative potentials .

• What are the specific characteristics of the MYF5 Antibody, FITC conjugated?

MYF5 Antibody, FITC conjugated is a polyclonal antibody typically derived from rabbit hosts that has been directly labeled with fluorescein isothiocyanate (FITC) . The antibody recognizes human MYF5 protein, with the immunogen being recombinant Human Myogenic factor 5 protein (amino acids 1-255) . This antibody preparation has several important technical specifications:

The FITC conjugation enables direct visualization of MYF5 protein without requiring secondary antibody staining, which simplifies experimental protocols and potentially reduces background signal .

• What is the subcellular localization of MYF5 and how does this impact detection methods?

MYF5 is predominantly localized to the nucleus, consistent with its function as a transcription factor that regulates muscle-specific gene expression . Immunofluorescence studies clearly demonstrate nuclear staining patterns in MYF5-expressing cells, including myoblasts and muscle stem cells . In C2C12 mouse myoblast cells, immunofluorescence with anti-MYF5 antibodies reveals specific nuclear staining, as confirmed by counterstaining with DAPI .

This nuclear localization has important methodological implications:

• Proper fixation and permeabilization protocols are essential to ensure antibody access to the nuclear compartment

• For flow cytometry applications, cells must be fixed with paraformaldehyde and permeabilized with agents like saponin

• The exclusive nuclear localization makes MYF5 an excellent marker for identifying nuclei of cells committed to the myogenic lineage

• Nuclear targeting of reporter proteins (as in the StemRep mouse model) facilitates quantification of MYF5-expressing cells in tissue sections

Understanding this subcellular localization is critical for optimizing detection protocols and interpreting experimental results when using MYF5 antibodies.

• How does MYF5 antibody specificity compare to other myogenic factor antibodies?

MYF5 shares significant sequence homology with other myogenic regulatory factors, particularly MyoD, which creates challenges for antibody specificity . Commercial MYF5 antibodies vary considerably in their specificity profiles, with some exhibiting cross-reactivity with MyoD and other related proteins . This cross-reactivity concern has led to the development of more specific monoclonal antibodies, including a rat monoclonal antibody specifically generated to overcome these limitations .

When evaluating MYF5 antibody specificity:

• Validation through immunoblot analysis enables identification of MYF5 protein from cell extracts

• Immunostaining should confirm proper nuclear localization pattern

• Comparison with known MYF5-expressing cell lines (like C2C12 myoblasts) provides positive controls

• Flow cytometry validation should include appropriate isotype controls

• Cross-reactivity testing against recombinant MyoD protein is advisable due to high sequence homology

Researchers should carefully validate each antibody lot before use in critical experiments to ensure specificity for MYF5 without cross-reactivity to related myogenic factors.

• What applications is the MYF5 antibody, FITC conjugated suitable for?

The MYF5 antibody, FITC conjugated, is validated for several research applications in muscle biology:

For flow cytometry applications, proper fixation with paraformaldehyde and permeabilization with saponin is essential for detecting this nuclear protein . The antibody has been successfully used to detect MYF5 in C2C12 mouse myoblast cells, demonstrating specific nuclear staining . While not explicitly mentioned for the FITC-conjugated version, related MYF5 antibodies have also been validated for Western blotting applications .

Advanced Research Applications

• How can FITC-conjugated MYF5 antibodies be used to distinguish between MYF5High and MYF5Low muscle stem cell populations?

FITC-conjugated MYF5 antibodies provide a powerful tool for identifying and isolating functionally distinct muscle stem cell (MuSC) subpopulations based on MYF5 expression levels . Flow cytometric analysis with these antibodies enables researchers to establish precise gating strategies that separate MYF5High and MYF5Low populations. These subpopulations have been shown to possess distinct biological characteristics:

For optimal detection, proper fixation (paraformaldehyde) and permeabilization (saponin) protocols are essential since MYF5 is a nuclear protein . Combined staining with PAX7 antibodies provides additional resolution for identifying specific subpopulations, revealing that MYF5Low cells represent a more uncommitted stem cell population .

• What are the optimal fixation and permeabilization protocols for detecting MYF5 using FITC-conjugated antibodies?

Detecting nuclear MYF5 requires carefully optimized fixation and permeabilization protocols to ensure antibody accessibility while preserving antigenic epitopes. Based on successful protocols reported in the literature:

For immunofluorescence microscopy on cultured cells:

• Fix cells by immersion in 4% paraformaldehyde for 10-15 minutes at room temperature

• Permeabilize with 0.1-0.5% Triton X-100 for 5-10 minutes

• Block with serum-containing buffer (5-10% serum from secondary antibody host species)

• Incubate with FITC-conjugated MYF5 antibody at optimized concentration (typically 5-10 μg/mL)

• Counterstain nuclei with DAPI or similar DNA dye

For flow cytometry applications:

• Fix cell suspension with paraformaldehyde

• Permeabilize with saponin

• Include isotype control (FITC-conjugated IgG from same host species)

• Establish compensation settings if performing multi-color analysis

Critical factors affecting staining quality include fixation duration, permeabilization agent concentration, antibody incubation time and temperature, and washing stringency. Researchers should optimize these parameters for their specific experimental system to achieve optimal signal-to-noise ratio.

• How can dual staining with MYF5 and PAX7 antibodies provide insights into muscle stem cell heterogeneity?

Dual immunostaining for MYF5 and PAX7 represents a powerful approach for investigating muscle stem cell heterogeneity and hierarchical relationships . PAX7 is a critical transcription factor maintaining the satellite cell pool, while MYF5 expression indicates commitment toward myogenic differentiation. Recent studies using dual reporter systems have revealed that muscle stem cells can be categorized into distinct subpopulations with different functional properties:

When implementing dual staining protocols, researchers should:

• Select antibodies from different host species (e.g., rabbit anti-MYF5 and mouse anti-PAX7)

• Use spectrally distinct fluorophores with minimal overlap

• Include appropriate single-stained and unstained controls

Analysis of muscle tissues from different physiological contexts using this approach has revealed age-related shifts in the MYF5High/MYF5Low ratio, with aged muscles showing proportionally more MYF5High cells, suggesting progressive loss of the undifferentiated stem cell pool during aging .

• What are the technical challenges in detecting low levels of MYF5 expression?

Detecting low levels of MYF5 expression presents several technical challenges that researchers must address:

• Signal strength limitations: Standard immunofluorescence may lack sensitivity to detect cells with low MYF5 expression, potentially leading to false negatives

• Background interference: Autofluorescence in the FITC channel (especially in tissue sections) can mask weak specific signals

• Fixation-related epitope masking: Overfixation may reduce antibody accessibility to nuclear MYF5 protein

• Transient expression dynamics: MYF5 levels fluctuate during myogenic progression, requiring precise timing of experiments

• Heterogeneous expression: Within a given sample, MYF5 expression can vary considerably between individual cells

To overcome these challenges, researchers can employ several strategies:

• Signal amplification techniques (tyramide signal amplification)

• Optimized fixation and permeabilization protocols specific for nuclear proteins

• Increased antibody concentration (with careful titration to avoid non-specific binding)

• Extended incubation times at lower temperatures

• Use of high-sensitivity detection systems

• Complementary analysis with mRNA detection methods

The StemRep reporter model, which enhances MYF5 detection through fluorescent protein tagging, provides one approach to overcoming these sensitivity limitations .

• How do MYF5 expression patterns change during muscle regeneration?

MYF5 expression undergoes dynamic changes during muscle regeneration, reflecting the sequential activation of genetic programs driving this process. Following injury, quiescent satellite cells are activated and transition from a predominantly PAX7+/MYF5Low state to PAX7+/MYF5High phenotype . This temporal pattern follows a specific sequence:

• Quiescent state: Satellite cells maintain low MYF5 levels with high PAX7 expression

• Early activation (24-48h post-injury): Rapid upregulation of MYF5 precedes MYOD expression

• Proliferation phase (48-72h): Continued high MYF5 expression with increasing MYOD levels

• Commitment to differentiation (72-96h): Some cells maintain MYF5 while downregulating PAX7

• Terminal differentiation: Eventual downregulation of MYF5 as cells mature into myofibers

• Self-renewal: A subset of activated cells returns to quiescence, reverting to PAX7+/MYF5Low state

Significantly, aged muscles show altered MYF5 expression dynamics, with:

• Higher baseline proportion of MYF5High cells in uninjured muscle

• Delayed but more prolonged MYF5 upregulation following injury

• Impaired return to the quiescent MYF5Low state

These changes in MYF5 expression patterns provide valuable insights into the molecular mechanisms governing muscle regeneration and age-related regenerative decline.

• What controls should be included when using MYF5 antibody, FITC conjugated for flow cytometry?

When utilizing FITC-conjugated MYF5 antibodies for flow cytometry, incorporating appropriate controls is essential for generating reliable and interpretable data:

Proper fixation with paraformaldehyde and permeabilization with saponin is essential for detecting nuclear MYF5 . When identifying MYF5High versus MYF5Low populations, carefully established gates based on control samples ensure accurate discrimination between these functionally distinct subpopulations .

• How can FITC-conjugated MYF5 antibodies be used in lineage tracing experiments?

FITC-conjugated MYF5 antibodies can be integrated into sophisticated lineage tracing experiments to investigate the developmental fate of MYF5-expressing cells:

• Isolation of distinct populations: MYF5High and MYF5Low cells can be isolated using FITC-conjugated MYF5 antibodies with fluorescence-activated cell sorting (FACS)

• In vitro fate mapping: Sorted populations can be cultured and monitored for:

  • Proliferation kinetics (comparing 634 cells/clone for MYF5High vs. 489 cells/clone for MYF5Low)

  • Differentiation capacity (MYF5High cells show 12-49% stronger MyoD induction)

  • Gene expression changes over time

• Combinatorial approaches: FITC-conjugated MYF5 antibodies can be used alongside:

  • Cell-tracing dyes for division tracking

  • Additional lineage markers (PAX7, MYOD, Myogenin) with spectrally distinct fluorophores

  • EdU/BrdU labeling for cell cycle analysis

• Transplantation experiments: Sorted MYF5High and MYF5Low populations can be transplanted into damaged muscle to assess regenerative capacity

The StemRep model provides a complementary genetic approach, using fluorescent reporter proteins to track MYF5 expression levels without antibody staining . Studies employing these approaches have revealed that MYF5Low cells retain greater stem cell characteristics while MYF5High cells progress more rapidly toward differentiation, providing important insights into myogenic lineage organization .