MYOF Antibody

What is the molecular structure and function of MYOF protein?

Myoferlin (MYOF), also known as FER1L3 and KIAA1207, belongs to the ferlin protein family. In humans, the canonical protein has 2061 amino acid residues with a molecular weight of approximately 234.7 kDa . MYOF plays critical roles in:

• Plasmalemma repair mechanisms in endothelial cells

• Endocytic recycling pathways

• VEGF signal transduction through regulation of KDR receptor levels

• Membrane dynamics in various cell types

Alternative splicing produces 8 different isoforms of this protein, and it is prominently expressed in myoblast and endothelial cells . At the subcellular level, MYOF is localized in the nucleus, cytoplasmic vesicles, and cell membrane .

How do I select the appropriate MYOF antibody for my specific application?

When selecting a MYOF antibody, consider these methodological factors:

• Target epitope location: Determine whether you need an antibody targeting a specific domain or region of MYOF

• Application compatibility: Verify the antibody has been validated for your specific application (WB, IHC, IF/ICC, Flow cytometry)

• Species reactivity: Ensure the antibody recognizes MYOF in your experimental species

• Antibody format: Choose between monoclonal, polyclonal, or recombinant antibodies based on your needs

For example, if studying MYOF in human samples via Western blot, an antibody like Proteintech's 19548-1-AP would be appropriate as it is validated for WB applications and shows reactivity with human samples at a recommended dilution of 1:1000-1:4000 .

What are the proper storage and handling procedures for MYOF antibodies?

To maintain antibody integrity and functionality:

• Long-term storage: Store at -20°C for up to one year

• Short-term/frequent use: Store at 4°C for up to one month

• Avoid freeze-thaw cycles: Repeated freezing and thawing significantly degrade antibody quality

• Consider aliquoting: For antibodies used frequently, create small aliquots to avoid repeated freeze-thaw cycles

• Buffer considerations: Most MYOF antibodies are stored in PBS with additives like sodium azide (0.02%) and glycerol (50%) at pH 7.3

When removing from storage, thaw antibodies on ice and centrifuge briefly to collect liquid at the bottom of the tube before use .

How can I validate the specificity of my MYOF antibody?

Antibody validation is critical for reproducible research. Implement these methodological approaches:

• Positive and negative controls:

  • Use cell lines known to express MYOF (positive controls: A549, HeLa, HepG2, placenta tissue)

  • Include a MYOF knockout cell line (e.g., MYOF knockout HeLa cell line) as a negative control

• Molecular weight verification:

  • Confirm the observed band matches the expected molecular weight (235-250 kDa for MYOF)

  • Note that MYOF may appear at multiple molecular weights (180 kDa and 250 kDa bands have been observed)

• Cross-validation with multiple antibodies:

  • Test at least two antibodies targeting different epitopes of MYOF

  • Compare results across different applications (WB, IF, etc.)

• Blocking peptide experiments:

  • Pre-incubate antibody with the immunizing peptide and compare with non-blocked antibody

A comprehensive validation should document: (i) that the antibody binds to the target protein; (ii) that it binds to the target protein in complex mixtures; (iii) that it doesn't bind to proteins other than the target; and (iv) that it performs as expected under specific experimental conditions .

What are the optimal conditions for Western blotting with MYOF antibodies?

Given the large size of MYOF (235 kDa), special considerations for Western blotting include:

• Gel preparation:

  • Use low percentage (6-8%) SDS-PAGE gels for better resolution of high molecular weight proteins

  • Consider gradient gels (4-12%) for improved separation

• Transfer conditions:

  • Employ wet transfer rather than semi-dry for large proteins

  • Extend transfer time (overnight at low voltage or 2-3 hours at higher voltage)

  • Add SDS (0.1%) to transfer buffer to improve large protein transfer

• Antibody incubation:

  • Primary antibody dilution: 1:1000-1:4000 for polyclonal antibodies , 1:2000-1:20000 for recombinant monoclonal antibodies

  • Blocking buffer: 5% non-fat dry milk in TBST is commonly used

  • Incubation time: Overnight at 4°C for primary antibody

• Detection optimization:

  • Exposure time varies significantly based on expression levels (3 seconds to 10 minutes)

  • For weakly expressed MYOF, longer exposure times or signal enhancers may be necessary

• Expected results:

  • MYOF typically appears as bands at 180 kDa and 250 kDa

  • Verify specificity using a MYOF knockout cell line as negative control

What strategies can resolve inconsistent immunostaining results with MYOF antibodies?

When troubleshooting immunostaining inconsistencies:

• Fixation optimization:

  • Test different fixatives: 100% methanol fixation has shown good results for MYOF detection

  • Compare paraformaldehyde (4%) versus methanol fixation methods

  • Fixation duration can significantly impact epitope accessibility

• Antigen retrieval enhancement:

  • For IHC applications, try TE buffer pH 9.0 for optimal retrieval

  • Alternative retrieval with citrate buffer pH 6.0 may be necessary for certain tissues

• Antibody titration:

  • Perform a dilution series (e.g., 1:125, 1:250, 1:500 for IF/ICC)

  • Determine optimal concentration for each tissue/cell type and fixation method

• Background reduction strategies:

  • If high background occurs due to Fc receptor binding, consider using F(ab) fragment secondary antibodies

  • Increase blocking time or concentration (5-10% normal serum from secondary antibody host species)

  • Include 0.3M glycine in blocking buffer to reduce non-specific protein interactions

• Secondary antibody selection:

  • Choose secondary antibodies that match both host species and isotype of primary MYOF antibody

  • For fluorescent detection, select appropriate fluorophores based on your microscope configuration

How should I interpret different banding patterns observed in Western blots with MYOF antibodies?

MYOF protein often presents complex banding patterns due to its large size, multiple isoforms, and post-translational modifications:

• Multiple band interpretation:

  • 235-250 kDa band: Full-length canonical MYOF protein

  • 180 kDa band: Potential alternative isoform or proteolytic fragment

  • Other bands: May represent splice variants (there are 8 known isoforms) or degradation products

• Cell/tissue-specific variations:

  • Expression levels and isoform distribution vary between cell types

  • Compare observed patterns with positive control cell lines (HeLa, A549, SK-BR-3)

• Verification approaches:

  • Use multiple antibodies targeting different MYOF epitopes

  • Compare results with genomic/transcriptomic data on MYOF isoform expression

  • Perform siRNA knockdown experiments to confirm specificity of observed bands

• Observed molecular weight discrepancies:

  • Post-translational modifications may cause migration shifts

  • Different running buffers and gel systems can affect apparent molecular weight

When publishing data, document the exact molecular weights observed, antibody used, and experimental conditions to facilitate reproducibility.

What approaches are recommended for designing antibodies against specific MYOF epitopes?

For researchers developing custom MYOF antibodies:

• Epitope selection principles:

  • Target unique, solvent-exposed regions of MYOF

  • Avoid highly conserved domains if species specificity is desired

  • Consider amino acid composition (avoid highly hydrophobic sequences)

  • Target regions distinct from other ferlin family members

• Computational design resources:

  • Utilize structure-based antibody engineering tools like OptCDR for designing complementarity-determining regions (CDRs)

  • Consider the three binding modes: lock and key, induced fit, and conformational selection

• Validation strategy planning:

  • Generate multiple antibody candidates against different epitopes

  • Include both N-terminal and C-terminal targeting antibodies

  • Design comprehensive validation experiments before production

• Testing recommendations:

  • Initial screening should include at least 20 designs per project

  • For redesign projects, consider multiple decoys and selective CDR redesign

  • Validate binding via multiple techniques (ELISA, BLI, SPR)

As noted in literature, "The complexity of optimizing several different antibody attributes using traditional immunization and screening methods has led to intense interest in developing antibody-design methods" .

How can discrepancies between different MYOF antibodies be reconciled in research studies?

When facing contradictory results from different MYOF antibodies:

• Epitope mapping analysis:

  • Determine the exact binding sites of each antibody

  • Consider epitope accessibility in different experimental conditions

  • Note whether antibodies target different MYOF domains that may be differentially exposed

• Methodological standardization:

  • Implement identical sample preparation protocols for all antibodies

  • Use consistent experimental conditions (buffers, incubation times, temperatures)

  • Process samples simultaneously when possible

• Comprehensive antibody validation:

  • Test each antibody against MYOF knockout controls

  • Perform peptide competition assays

  • Consider orthogonal validation methods (mass spectrometry)

• Isoform-specific considerations:

  • Determine if discrepancies arise from isoform-specific detection

  • Map antibody epitopes to specific MYOF isoforms

  • Use RT-PCR to correlate isoform expression with antibody detection patterns

• Data integration strategies:

  • Report results from multiple antibodies with transparent documentation of differences

  • Use orthogonal techniques (qPCR, mass spectrometry) to resolve conflicts

  • Consider creating a consensus result based on multiple antibodies

"When using antibodies in an experiment, the characterization of the antibody needs to document... that the antibody is binding to the target protein... that the antibody binds to the target protein when in a complex mixture of proteins... that the antibody does not bind to proteins other than the target protein" .

What are the considerations for using MYOF antibodies in multiplex immunofluorescence studies?

For successful multiplex detection involving MYOF:

• Antibody panel design:

  • Select MYOF antibodies from different host species than other target antibodies

  • Consider using directly conjugated antibodies to simplify detection

  • Test for cross-reactivity between all antibodies in the panel

• Sequential staining protocol development:

  • Determine optimal staining sequence (typically start with lowest abundance target)

  • Implement complete washing steps between antibody applications

  • Consider signal stripping or quenching for sequential protocols

• Spectral considerations:

  • Choose fluorophores with minimal spectral overlap

  • Include proper controls for spectral unmixing

  • Account for tissue autofluorescence, particularly in muscle samples

• Validation requirements:

  • Test each antibody individually before multiplexing

  • Include single-stained controls alongside multiplex samples

  • Confirm pattern consistency between single and multiplex staining

• Image acquisition optimization:

  • Adjust exposure times for each channel independently

  • Consider sequential rather than simultaneous acquisition

  • Implement appropriate background subtraction methods

How can MYOF antibodies be employed for studying exosome biology and intercellular communication?

MYOF's role in membrane dynamics makes it relevant for exosome research:

• Exosome isolation and characterization:

  • Use MYOF antibodies to assess exosome membrane composition

  • Implement immunoprecipitation techniques to isolate MYOF-containing exosomes

  • Compare MYOF content across exosomes from different cell types

• Optimization for small vesicle detection:

  • Use super-resolution microscopy to visualize MYOF on individual exosomes

  • Consider immunogold labeling for electron microscopy detection

  • Implement flow cytometry with beads for exosome-bound MYOF detection

• Functional studies methodology:

  • Track MYOF-containing exosomes in recipient cells using labeled antibodies

  • Employ MYOF antibodies to block potential functional domains on exosomes

  • Use proximity ligation assays to study MYOF interactions with other proteins

• Technical considerations:

  • Minimize background through careful blocking and antibody dilution

  • Include detergent controls to distinguish membrane-bound from luminal MYOF

  • Validate antibody specificity in the context of small vesicles

This application represents an advanced use of MYOF antibodies that requires careful optimization and comprehensive controls.