MYRF Antibody

What is MYRF and why is it important to study with antibody-based approaches?

MYRF (myelin regulatory factor) is a membrane-bound transcription factor that plays critical roles in multiple biological processes, including oligodendrocyte differentiation, embryonic development, molting, and synaptic plasticity. The protein is approximately 124.4 kilodaltons in mass and may also be referred to as MRF, Ndt80, 11orf9, C11orf9, CUGS, or MMERV in the literature .

What makes MYRF particularly interesting is its unusual mechanism of activation. MYRF undergoes autolytic cleavage, after which its N-terminal fragment translocates to the nucleus as a homo-trimer to function as a transcription factor . This homo-trimerization is essential for its DNA-binding specificity and transcriptional activity. Antibody-based detection methods are crucial for studying this process because they allow researchers to track both the full-length membrane-bound form and the cleaved nuclear form of the protein.

For effective study of MYRF, researchers should select antibodies that recognize specific domains (N-terminal or C-terminal) depending on whether they wish to study the nuclear transcription factor fragment or the membrane-bound full-length protein.

What are the primary applications for MYRF antibodies in neural research?

MYRF antibodies have several critical applications in neural research, particularly in studying oligodendrocyte biology and myelination processes. The most common applications include:

Western Blotting: Allows detection of both the full-length (~140 kDa) and cleaved N-terminal fragment (~70 kDa) of MYRF, enabling researchers to assess processing efficiency and protein expression levels . This application is supported by multiple commercially available antibodies with human, mouse, and rat reactivity .

Immunohistochemistry/Immunofluorescence: Enables visualization of MYRF protein localization in tissue sections or cultured cells, which is particularly useful for tracking nuclear translocation after cleavage. Both paraffin-embedded (IHC-p) and frozen section (IHC-fr) compatible antibodies are available .

Co-immunoprecipitation: Critical for studying MYRF protein-protein interactions, particularly homo-trimerization and interactions with other transcription factors. This approach has been instrumental in understanding how mutations affect MYRF complex formation .

Chromatin Immunoprecipitation: Used to identify MYRF binding sites in the genome, helping to elucidate its direct transcriptional targets in oligodendrocytes and other cell types.

For neural research specifically, it's important to select antibodies validated in relevant neural cell types, particularly oligodendrocyte precursor cells and mature oligodendrocytes, where MYRF plays a critical regulatory role.

How do I select the appropriate MYRF antibody for my research question?

Selecting the appropriate MYRF antibody requires careful consideration of multiple factors to ensure experimental success:

Epitope location: Determine whether you need to detect the N-terminal (transcription factor) domain or the C-terminal domain. For studying nuclear translocation, antibodies recognizing the N-terminal region are preferable. The search results show multiple antibodies targeting different regions, including N-terminal specific options .

Species reactivity: Verify cross-reactivity with your experimental model. Available antibodies show reactivity with human, mouse, rat, and other species including canine, guinea pig, and zebrafish samples . If working with less common models, validate the antibody specifically in your species of interest.

Application compatibility: Ensure the antibody is validated for your intended application. The available MYRF antibodies vary in their validated applications, with some optimized for Western blot only, while others are validated for multiple applications including WB, IHC, IF, and ELISA .

Conjugation requirements: Consider whether you need unconjugated antibodies (most common) or conjugated versions (biotin, RBITC, Cy5) for specialized applications. Conjugated antibodies may be particularly useful for multi-color immunofluorescence studies .

Validation data: Review antibody validation data, particularly citations in peer-reviewed literature. Several antibodies in the search results include citation information, which can provide confidence in their performance in specific applications .

For complex experiments studying MYRF processing or trimerization, consider using combinations of antibodies recognizing different domains to comprehensively track MYRF biology.

What controls are essential when using MYRF antibodies in experimental settings?

Implementing proper controls is critical for ensuring the validity of experiments using MYRF antibodies. The following controls should be considered:

Positive controls: Include samples known to express MYRF, such as differentiating oligodendrocytes or cells transfected with MYRF expression constructs. The search results mention Oli-neu cell lines with doxycycline-inducible MYRF expression that can serve as excellent positive controls .

Negative controls: Use samples where MYRF expression is absent or knocked down. Additionally, non-cleaving mutants like MYRF-K592A can serve as controls for experiments focused on the processed form of MYRF .

Antibody validation controls:

• Primary antibody omission

• Isotype controls

• Blocking peptide competition

• siRNA/shRNA knockdown of MYRF

Functional mutant controls: The search results describe several MYRF mutants that can serve as functional controls, including:

• MYRF-K592A: A mutant that does not undergo auto-cleavage

• MYRF-R454A: A mutant that does not bind to DNA

• Various DNA-binding domain mutants (F387S, Q403H, G435R, L479V): These affect trimerization and can serve as controls in oligomerization studies

For co-immunoprecipitation experiments studying MYRF trimerization, the G566R mutation (equivalent to human G435R) provides an excellent negative control, as it fails to form homo-trimers despite undergoing proper cleavage .

How can I optimize MYRF antibody performance in Western blot applications?

Optimizing MYRF antibody performance in Western blot applications requires attention to several technical factors:

Sample preparation considerations:

• Include protease inhibitors to prevent degradation of MYRF protein

• Use appropriate lysis buffers to extract both membrane-bound and nuclear forms

• Consider subcellular fractionation to separately analyze membrane and nuclear fractions

Gel selection and transfer parameters:

• Use lower percentage gels (6-8%) for full-length MYRF (~140 kDa)

• Consider gradient gels when analyzing both full-length and cleaved fragments simultaneously

• Transfer large proteins at lower voltage for longer time or use wet transfer systems

Antibody incubation optimization:

• Test a range of antibody dilutions (typically 1:500 to 1:2000 based on commercial recommendations)

• Optimize primary antibody incubation time and temperature

• Consider using milk-based blocking for some antibodies and BSA-based blocking for others

Visualization techniques:

• For studying MYRF processing, chemiluminescence with longer exposure times may be necessary to capture both strong and weak signals

• For quantitative analysis, consider fluorescent secondary antibodies for more precise quantification

Molecular weight expectations:
When analyzing MYRF by Western blot, expect the following species:

• Full-length MYRF: ~140 kDa

• N-terminal fragment (post-cleavage): ~70 kDa

• N-terminal trimer (under non-reducing conditions): ~210 kDa

The research data indicates that mutations in the DNA-binding domain affect the migration pattern of MYRF N-terminal fragments, with mutants appearing as monomers rather than trimers on non-reducing gels .

What are the critical methodological considerations for using MYRF antibodies in immunohistochemistry?

When using MYRF antibodies for immunohistochemistry (IHC), several methodological factors must be optimized:

Tissue preparation and fixation:

• For paraffin-embedded sections: 4% paraformaldehyde fixation followed by proper antigen retrieval (typically heat-mediated citrate buffer) is recommended

• For frozen sections: Light fixation (2-4% PFA for 10-20 minutes) often preserves MYRF antigenicity better

Antibody selection:

• Verify IHC validation data for your specific antibody

• Select antibodies specifically validated for IHC-p or IHC-fr based on your sample type

• Consider using antibodies that recognize different domains for comprehensive analysis

Signal amplification:

• Tyramide signal amplification may be helpful for detecting low MYRF expression

• Biotin-conjugated MYRF antibodies can be used with streptavidin-based detection systems

Co-localization studies:

• For studying oligodendrocyte development, co-stain with markers like Olig2, Sox10, or CC1

• Use confocal microscopy to accurately determine nuclear localization of cleaved MYRF

Quantification approaches:

• For developmental studies, quantify the percentage of cells showing nuclear MYRF localization

• In disease models, assess changes in MYRF subcellular distribution

Remember that MYRF undergoes a transition from cytoplasmic/membrane localization to nuclear localization upon cleavage, so proper interpretation of staining patterns is critical. Cellular context and developmental stage should always inform the expected localization pattern.

How can MYRF antibodies be used to investigate the mechanism of MYRF auto-processing and trimerization?

Investigating MYRF auto-processing and trimerization requires sophisticated experimental approaches using specific antibodies:

Co-immunoprecipitation for trimerization studies:
The search results describe co-immunoprecipitation experiments to assess MYRF homo-trimerization . This approach can be adapted by:

• Using differently tagged MYRF constructs (e.g., Flag-tagged and HA-tagged)

• Immunoprecipitating with one tag and blotting with antibodies against the other

• Comparing wild-type MYRF with trimerization-deficient mutants (F387S, Q403H, G435R, L479V)

Non-reducing gel electrophoresis:
The search results indicate that MYRF trimerization can be visualized on non-reducing gels, where wild-type N-terminal fragments appear as trimers (~210 kDa) while mutants appear as monomers (~70 kDa) . This technique requires:

• Sample preparation without reducing agents

• Careful temperature control to maintain oligomeric states

• Antibodies that recognize epitopes accessible in both monomeric and trimeric forms

Pulse-chase analysis of MYRF processing:

• Metabolic labeling with radiolabeled amino acids

• Immunoprecipitation at various time points

• Analysis of the conversion of full-length to cleaved forms

Subcellular fractionation:

• Separate membrane, cytosolic, and nuclear fractions

• Analyze each fraction by Western blot using MYRF antibodies

• Quantify the distribution of full-length and cleaved forms

Proximity ligation assays:

• Use pairs of antibodies recognizing different epitopes on MYRF

• Detect and quantify trimerization in situ through fluorescent signals generated only when epitopes are in close proximity

For studying auto-processing specifically, comparing wild-type MYRF with the non-cleaving K592A mutant provides valuable insights into the cleavage mechanism and its cellular consequences .

What approaches can be used to study the impact of MYRF mutations on its DNA-binding and transcriptional activity?

The search results describe several methodological approaches for studying how mutations affect MYRF function :

Luciferase reporter assays:
The described research used a reporter construct containing MYRF binding sites from the Rffl locus cloned into pGL3 promoter vector . Key considerations include:

• Using low DNA amounts (2 ng) to minimize overexpression artifacts

• Including appropriate controls (empty vector, non-cleaving K592A mutant, DNA-binding deficient R454A mutant)

• Normalizing reporter activity to account for transfection efficiency

Gene expression analysis in inducible cell lines:
The research utilized Oli-neu cell lines with doxycycline-inducible expression of wild-type and mutant MYRF . This approach offers several advantages:

• Controlled expression to avoid toxicity issues

• Ability to induce MYRF at specific time points

• Comparison of multiple mutants under identical conditions

Chromatin immunoprecipitation (ChIP):

• Use MYRF antibodies to immunoprecipitate chromatin

• Compare binding of wild-type and mutant MYRF to known target sites

• Combine with sequencing (ChIP-seq) for genome-wide binding analysis

Electrophoretic mobility shift assays (EMSA):

• Use recombinant MYRF DNA-binding domains

• Compare DNA-binding properties of wild-type and mutant proteins

• Assess binding specificity and affinity for different DNA sequences

Protein-DNA binding kinetics:

• Surface plasmon resonance or bio-layer interferometry

• Measure association and dissociation rates

• Determine how mutations affect binding affinity and stability

The research demonstrates that mutations in the MYRF DNA-binding domain (F387S, Q403H, G435R, L479V) severely impair transcriptional activity, primarily by disrupting homo-trimerization which is essential for proper DNA binding .

How can researchers investigate the role of MYRF in oligodendrocyte differentiation and myelination processes?

Investigating MYRF's role in oligodendrocyte biology requires integrated approaches combining antibody-based techniques with functional assays:

Temporal expression analysis:

• Use MYRF antibodies to track protein expression during oligodendrocyte differentiation

• Perform Western blot and immunocytochemistry at multiple time points

• Correlate MYRF nuclear localization with expression of myelin genes

Conditional knockout/knockdown studies:

• Generate oligodendrocyte-specific MYRF knockout models

• Use immunohistochemistry with MYRF antibodies to confirm deletion

• Assess consequences on myelination using myelin protein antibodies (MBP, PLP)

Rescue experiments:

• Reintroduce wild-type or mutant MYRF into knockout backgrounds

• Use antibodies to confirm expression and proper localization

• Assess functional recovery of myelination capacity

Chromatin dynamics:

• Combine MYRF ChIP-seq with histone modification ChIP-seq

• Investigate how MYRF binding influences chromatin accessibility

• Use MYRF antibodies for sequential ChIP to identify co-binding partners

Single-cell analysis:

• Perform single-cell immunofluorescence to correlate MYRF nuclear localization with differentiation state

• Combine with RNA-scope to assess target gene activation at the single-cell level

The search results indicate that MYRF is essential for oligodendrocyte differentiation, and its function depends on proper trimerization after autolytic cleavage . The Oli-neu cell line system described provides a valuable model for studying how MYRF mutations affect this process .

What are the technical challenges in detecting both membrane-bound and cleaved forms of MYRF simultaneously?

Detecting both membrane-bound and cleaved forms of MYRF presents several technical challenges that require careful experimental design:

Antibody epitope considerations:

• Use antibodies recognizing the N-terminal domain to detect both full-length and cleaved nuclear forms

• Consider using paired antibodies against N-terminal and C-terminal domains for comprehensive analysis

• Validate antibody specificity against both forms using appropriate controls

Sample preparation challenges:

• Standard lysis protocols may favor detection of either membrane or nuclear proteins

• Use fractionation approaches to optimize extraction of both pools

• Consider specialized detergents for membrane protein solubilization (e.g., NP-40, Triton X-100)

Immunofluorescence visualization:

• Membrane-bound MYRF may give weak, diffuse signals compared to concentrated nuclear signals

• Use confocal microscopy with Z-stack imaging to distinguish membrane from intracellular staining

• Consider super-resolution microscopy for detailed subcellular localization

Western blot optimization:

• The large size difference between full-length (~140 kDa) and cleaved (~70 kDa) forms requires gradient gels or separate optimized blots

• Transfer efficiency may differ between large and small proteins

• Different exposure times may be needed to visualize both forms optimally

Quantification challenges:

• Develop standardized approaches to quantify the ratio of cleaved to uncleaved forms

• Use purified recombinant proteins as standards for absolute quantification

• Consider using fluorescent secondary antibodies for more precise quantification

A methodological table for detecting different MYRF forms:

The research indicates that mutations in the DNA-binding domain affect both trimerization and in some cases (G435R) the efficiency of auto-cleavage , highlighting the importance of monitoring both processes.

What are the emerging applications of MYRF antibodies in disease research?

MYRF antibodies are increasingly being used to study disease processes beyond their established role in developmental biology:

Demyelinating disorders:

• Monitoring MYRF expression and function in multiple sclerosis models

• Assessing remyelination potential by tracking MYRF activation in oligodendrocyte precursor cells

• Correlating MYRF activity with remyelination efficiency in different disease stages

Neurodevelopmental disorders:

• Investigating MYRF mutations identified in congenital disorders

• Studying how the DNA-binding domain mutations (F387S, Q403H, G435R, L479V) impact brain development

• Using MYRF antibodies as markers for proper oligodendrocyte differentiation in developmental models

Cancer biology:

• Exploring MYRF's potential role in glioma development and progression

• Using MYRF antibodies to classify central nervous system tumors

• Investigating potential correlations between MYRF expression and tumor aggressiveness

Therapeutic development:

• Screening compounds that enhance MYRF expression or nuclear translocation

• Using MYRF antibodies to monitor treatment effects on oligodendrocyte differentiation

• Developing high-throughput immunoassays for drug screening

As research progresses, MYRF antibodies will likely become important tools for understanding the molecular mechanisms underlying various disorders and for developing targeted therapeutic approaches aimed at modulating MYRF function.

What methodological advances may improve MYRF antibody applications in future research?

Several technological advances promise to enhance the utility of MYRF antibodies in research:

Single-cell proteomics:

• Combining MYRF antibodies with mass cytometry (CyTOF) for single-cell protein analysis

• Correlating MYRF expression with multiple other markers in heterogeneous cell populations

• Developing antibody panels specifically optimized for oligodendrocyte lineage analysis

Live-cell imaging approaches:

• Developing membrane-permeable MYRF antibody fragments for live imaging

• Creating nanobodies against MYRF for improved access to subcellular compartments

• Using split fluorescent protein complementation to visualize MYRF trimerization in living cells

Advanced microscopy methods:

• Implementing expansion microscopy to improve spatial resolution of MYRF localization

• Using lattice light-sheet microscopy for dynamic studies of MYRF processing

• Applying correlative light and electron microscopy to connect MYRF localization with ultrastructure

Improved recombinant antibodies:

• Developing monoclonal recombinant antibodies with enhanced specificity for different MYRF domains

• Creating conformation-specific antibodies that selectively recognize trimeric MYRF

• Engineering antibodies that specifically detect cleaved vs. uncleaved forms

Combination with CRISPR technologies:

• Using CRISPR-mediated tagging of endogenous MYRF for improved antibody detection

• Developing CRISPR screening approaches to identify regulators of MYRF processing

• Creating knock-in reporter lines to complement antibody-based detection

These methodological advances will enable more precise characterization of MYRF biology and its role in both normal development and disease processes, ultimately contributing to a deeper understanding of myelination and potentially to new therapeutic approaches for myelin-related disorders.