MYEF2 Antibody

What is MYEF2 and what are its primary biological functions?

MYEF2 (Myelin Expression Factor 2) functions primarily as a transcriptional repressor of the myelin basic protein gene (MBP). It specifically binds to the proximal MB1 element 5'-TTGTCC-3' of the MBP promoter. Research indicates that its binding to MB1 and subsequent function are inhibited by PURA protein (by similarity) . MYEF2 is predominantly localized in the nucleus, consistent with its role in transcriptional regulation . Recent studies have identified MYEF2 as potentially significant in various cancers, particularly hepatocellular carcinoma (HCC), where its expression is upregulated compared to normal tissues .

What applications are MYEF2 antibodies commonly used for in research?

MYEF2 antibodies are primarily utilized in Western Blotting (WB), Immunohistochemistry (IHC), and Immunofluorescence (IF) applications . These applications enable researchers to:

• Detect and quantify MYEF2 protein expression in tissue samples and cell lines

• Visualize subcellular localization (predominantly nuclear)

• Examine expression patterns in pathological versus normal tissues

• Validate knockdown or overexpression efficiency in functional studies

Verified samples for Western blotting include A172 and TM4 cell lines, while verified samples for immunohistochemistry include human esophageal cancer and liver cancer tissues .

What factors should be considered when selecting a MYEF2 antibody for specific applications?

When selecting a MYEF2 antibody, researchers should consider:

• Target epitope region: Antibodies targeting different regions (N-terminal, middle region, C-terminal) may yield different results depending on protein conformation or post-translational modifications

• Host species: Most available MYEF2 antibodies are rabbit polyclonal antibodies

• Cross-reactivity: Some antibodies demonstrate broad species reactivity (human, mouse, rat, dog, cow, etc.), while others have more limited reactivity

• Validated applications: Ensure the antibody has been validated for your specific application (WB, IHC, IF)

• Clonality: Polyclonal antibodies provide broader epitope recognition but may have batch-to-batch variations

For optimal results, antibodies should be tested at different dilutions within the recommended range (typically 1:500-1:2000 for WB and 1:50-1:200 for IHC) .

How should researchers optimize Western blotting protocols for MYEF2 detection?

For optimal MYEF2 detection via Western blotting:

• Protein extraction: Use nuclear extraction protocols as MYEF2 is primarily nuclear-localized

• Loading controls: Include appropriate nuclear protein loading controls

• Dilution optimization: Test dilutions within the 1:500-1:2000 range to determine optimal signal-to-noise ratio

• Molecular weight considerations: While the calculated molecular weight of MYEF2 is approximately 64 kDa, the observed band may differ from expected size due to post-translational modifications

• Blocking conditions: Use 5% non-fat milk or BSA in TBST for blocking to minimize background

• Incubation time and temperature: Optimize primary antibody incubation (typically overnight at 4°C)

• Multiple band interpretation: Be aware that multiple bands may appear if different modified forms of the protein are present simultaneously

When troubleshooting unexpected band patterns, consider post-translational modifications that may affect protein mobility on SDS-PAGE.

What are the critical steps for successful immunohistochemistry staining with MYEF2 antibodies?

For robust immunohistochemistry results with MYEF2 antibodies:

• Tissue fixation and processing: Use appropriate fixation methods (typically 10% neutral buffered formalin) and paraffin embedding

• Antigen retrieval: Optimize antigen retrieval methods (heat-induced epitope retrieval in citrate buffer pH 6.0 or EDTA buffer pH 9.0)

• Blocking endogenous peroxidase: Use 3% hydrogen peroxide for 10 minutes to block endogenous peroxidase activity

• Antibody dilution: Test dilutions between 1:50-1:200 to determine optimal concentration

• Positive controls: Include verified positive controls such as human esophageal cancer or liver cancer tissues

• Negative controls: Include antibody diluent-only controls to assess background staining

• Counterstaining: Use appropriate nuclear counterstain (e.g., hematoxylin) to visualize cellular context

• Subcellular localization assessment: Confirm nuclear localization pattern consistent with MYEF2 function

Remember that MYEF2 staining should show predominantly nuclear localization as observed in THE HUMAN PROTEIN ATLAS data .

How can researchers effectively validate MYEF2 antibody specificity?

To validate MYEF2 antibody specificity:

• Peptide competition assay: Pre-incubate antibody with immunizing peptide before application to confirm specific binding

• Knockdown validation: Perform siRNA knockdown of MYEF2 and confirm reduced signal intensity by Western blot

• Overexpression validation: Transfect cells with MYEF2 expression plasmids and confirm increased signal intensity

• Multiple antibody comparison: Use antibodies targeting different epitopes of MYEF2 to confirm consistent localization and expression patterns

• Cross-species validation: Test antibody in species with high sequence homology to confirm expected patterns

• Multiple technique validation: Confirm findings using complementary techniques (e.g., verify WB results with IHC or IF)

Researchers have successfully validated MYEF2 antibody specificity using siRNA knockdown in SK-HEP-1 and Hep 3B2.1-7 cells, as well as plasmid-based overexpression in PLC/PRF/5 cells .

How should researchers interpret variable MYEF2 banding patterns in Western blots?

When analyzing Western blot results for MYEF2:

• Multiple bands interpretation: The observed molecular weight of MYEF2 may differ from the calculated 64 kDa due to:

  • Post-translational modifications (phosphorylation, glycosylation)

  • Alternative splicing variants

  • Proteolytic processing

  • Protein-protein interactions affecting mobility

• Tissue/cell-specific variations: Different cell types may express different MYEF2 isoforms or post-translationally modified variants

• Experimental conditions affecting band patterns:

  • Sample preparation methods (denaturing vs. native conditions)

  • Buffer composition

  • Gel percentage

  • Running conditions

• Verification strategies:

  • Use multiple antibodies targeting different epitopes

  • Compare with recombinant MYEF2 protein controls

  • Perform phosphatase treatment to identify phosphorylation-dependent bands

The observation that "the actual band is not consistent with the expectation" is a documented phenomenon with MYEF2 antibodies , likely due to these factors.

What controls should be included when studying MYEF2 in pathological conditions?

When studying MYEF2 in disease contexts, include:

• Tissue/cell controls:

  • Matched normal adjacent tissue from the same patient

  • Healthy tissue from non-diseased individuals

  • Cell lines with known MYEF2 expression levels (A172, TM4)

• Technical controls:

  • Isotype controls to assess non-specific binding

  • Secondary antibody-only controls

  • Blocking peptide controls

• Expression validation controls:

  • mRNA expression correlation (qPCR)

  • Multiple antibodies targeting different epitopes

  • Positive control tissues (human esophagus cancer, human liver cancer)

• Functional controls:

  • MYEF2 knockdown cells

  • MYEF2 overexpression cells

  • Transcriptional activity assays (for repressor function)

In HCC studies, researchers should consider comparing MYEF2 expression across different tumor stages, grades, and patient survival outcomes as demonstrated in The Cancer Genome Atlas analyses .

How can MYEF2 antibodies be utilized to investigate its role in transcriptional regulation?

To study MYEF2's transcriptional repressor function:

• Chromatin Immunoprecipitation (ChIP):

  • Use MYEF2 antibodies to immunoprecipitate DNA-protein complexes

  • Analyze binding to MB1 element (5'-TTGTCC-3') in the MBP promoter

  • Identify novel genomic binding sites through ChIP-seq

• Co-immunoprecipitation (Co-IP):

  • Identify protein interaction partners using MYEF2 antibodies

  • Investigate the relationship with PURA, which inhibits MYEF2 binding and function

• Luciferase reporter assays:

  • Construct reporters containing MBP promoter elements

  • Measure transcriptional repression upon MYEF2 overexpression

  • Assess the impact of mutations in the MB1 element

• Immunofluorescence co-localization:

  • Examine nuclear co-localization with transcriptional machinery components

  • Visualize dynamic changes in localization during cell differentiation or stress conditions

• Proximity ligation assays:

  • Detect and quantify interactions between MYEF2 and other transcriptional regulators in situ

These approaches can help elucidate the molecular mechanisms underlying MYEF2's role as a transcriptional repressor.

What methods can be used to study MYEF2 post-translational modifications using antibodies?

To investigate MYEF2 post-translational modifications:

• Phospho-specific antibodies:

  • Use antibodies targeting specific phosphorylation sites (pThr319, pSer408, Ser890)

  • Compare phosphorylation status across different cellular conditions or disease states

• 2D gel electrophoresis with Western blotting:

  • Separate proteins by both isoelectric point and molecular weight

  • Detect MYEF2 isoforms with different modification patterns

• Phosphatase treatment:

  • Treat samples with phosphatases before Western blotting

  • Compare band patterns before and after treatment

• Mass spectrometry validation:

  • Immunoprecipitate MYEF2 using specific antibodies

  • Analyze post-translational modifications by mass spectrometry

• Functional correlation studies:

  • Correlate phosphorylation status with MYEF2 transcriptional repressor activity

  • Examine the impact of kinase inhibitors on MYEF2 function and localization

The availability of phospho-specific MYEF2 antibodies (pThr319, pSer408, Ser890) enables detailed investigation of how phosphorylation regulates MYEF2 function.

How can researchers integrate MYEF2 antibody-based detection with functional studies in cancer research?

For comprehensive investigation of MYEF2 in cancer:

• Expression correlation with clinicopathological features:

  • Use IHC to analyze MYEF2 expression across different cancer stages and grades

  • Correlate expression with patient survival data

  • Develop scoring systems based on staining intensity and distribution

• Functional validation through genetic manipulation:

  • Perform siRNA knockdown (as demonstrated in SK-HEP-1 and Hep 3B2.1-7 cells)

  • Create overexpression models (as shown in PLC/PRF/5 cells)

  • Validate knockdown/overexpression efficiency using antibody-based detection (WB, IHC)

• Phenotypic assays with molecular correlation:

  • Assess changes in invasion and migration (transwell and wound healing assays)

  • Correlate with molecular pathways using antibody panels

  • Investigate downstream targets of MYEF2 transcriptional regulation

• Therapeutic response prediction:

  • Evaluate MYEF2 expression before and after treatment

  • Correlate expression patterns with treatment response

  • Develop predictive models integrating MYEF2 with other biomarkers

In HCC research, MYEF2 expression correlates with advanced disease stage (stage 2-4), poor differentiation (G3-G4), and worse survival outcomes (OS and DSS), suggesting its potential as a prognostic biomarker .

What considerations should researchers take when designing immunofluorescence experiments with MYEF2 antibodies?

For optimal immunofluorescence studies:

• Fixation method optimization:

  • Compare paraformaldehyde, methanol, and acetone fixation

  • Optimize fixation time to preserve epitope accessibility while maintaining cellular architecture

• Permeabilization conditions:

  • Test different permeabilization agents (Triton X-100, saponin)

  • Optimize concentration and duration to ensure nuclear penetration

• Co-localization studies:

  • Pair MYEF2 antibodies with markers of nuclear compartments

  • Use confocal microscopy for precise localization analysis

• Signal amplification:

  • Consider tyramide signal amplification for low-abundance detection

  • Optimize antibody concentration to maximize signal-to-noise ratio

• Spectral considerations:

  • Choose fluorophores with minimal spectral overlap when multiplexing

  • Include single-color controls for spectral unmixing

• Image acquisition settings:

  • Standardize exposure settings across experimental conditions

  • Use Z-stacking to capture the full nuclear volume

  • Apply deconvolution for improved resolution

• Quantitative analysis:

  • Develop consistent methods for quantifying nuclear MYEF2 intensity

  • Correlate intensity with functional outcomes

Researchers should note that MYEF2 displays predominantly nuclear localization, consistent with its function as a transcriptional regulator .

What is the significance of MYEF2 expression patterns in hepatocellular carcinoma?

Research findings on MYEF2 in HCC reveal:

These findings suggest MYEF2 may serve as both a diagnostic and prognostic biomarker for HCC, with potential implications for personalized treatment approaches.

How can researchers address contradictory data regarding MYEF2 expression across different cancer types?

To resolve contradictory findings:

• Methodological standardization:

  • Use standardized antibody validation protocols

  • Implement consistent scoring systems for expression analysis

  • Apply uniform sample processing procedures

• Context-dependent analysis:

  • Acknowledge tissue-specific roles of MYEF2

  • Consider microenvironmental factors influencing expression

  • Evaluate expression in relation to cellular differentiation state

• Integrated multi-omics approach:

  • Correlate protein expression (antibody-based) with mRNA levels

  • Incorporate mutation and copy number analysis

  • Consider epigenetic regulation mechanisms

• Biological validation:

  • Perform functional studies in multiple cell lines

  • Use in vivo models to validate in vitro findings

  • Apply CRISPR/Cas9 technology for precise genetic manipulation

• Meta-analysis frameworks:

  • Systematically review existing literature

  • Apply statistical methods to address heterogeneity

  • Consider publication bias in data interpretation

While ONCOMINE data shows MYEF2 upregulation in colorectal cancer, leukemia, liver cancer, melanoma, and ovarian cancer, it also shows downregulation in other cancer types , highlighting the importance of context-specific analysis.

What emerging applications exist for MYEF2 antibodies in single-cell analysis and spatial transcriptomics?

Emerging applications include:

• Single-cell protein analysis:

  • Mass cytometry (CyTOF) incorporating MYEF2 antibodies

  • Single-cell Western blotting for heterogeneity assessment

  • Microfluidic antibody capture for protein quantification

• Spatial proteomics:

  • Multiplexed immunofluorescence with MYEF2 antibodies

  • Imaging mass cytometry for spatial protein mapping

  • Co-detection by indexing (CODEX) for multiplexed protein visualization

• Integrated multi-modal analysis:

  • Paired single-cell transcriptomics and proteomics

  • Spatial transcriptomics with protein validation

  • In situ sequencing with protein co-detection

• Computational integration:

  • Algorithms for correlating spatial protein and RNA data

  • Machine learning approaches for pattern recognition

  • Trajectory analysis incorporating MYEF2 expression dynamics

• Functional spatial biology:

  • Photactivatable antibodies for region-specific manipulation

  • Optogenetic control coupled with MYEF2 detection

  • Spatial analysis of transcription factor networks

These approaches could reveal previously unrecognized heterogeneity in MYEF2 expression and function within tumors, potentially identifying specialized cellular niches or transition states relevant to cancer progression.

What are common challenges in MYEF2 antibody applications and how can they be addressed?

How should researchers validate experimental findings when discrepancies emerge between MYEF2 antibody-based detection methods?

When facing methodological discrepancies:

• Orthogonal validation approach:

  • Compare protein detection (antibody-based) with mRNA analysis (qPCR)

  • Validate findings using multiple antibodies targeting different epitopes

  • Confirm results with alternative detection technologies (mass spectrometry)

• Technical validation:

  • Implement standardized positive and negative controls

  • Perform replicate experiments under identical conditions

  • Use alternative sample preparation methods

• Biological validation:

  • Assess MYEF2 knockdown and overexpression models

  • Correlate with functional outcomes (transcriptional regulation)

  • Evaluate in multiple cell lines or tissue types

• Quantitative assessment:

  • Apply objective scoring systems for IHC

  • Use digital image analysis for standardized quantification

  • Implement statistical approaches to evaluate significance of differences

• Metadata documentation:

  • Record detailed experimental conditions

  • Document antibody information (lot number, age, storage conditions)

  • Maintain comprehensive protocol records

What storage and handling practices maximize MYEF2 antibody performance and longevity?

To optimize antibody performance:

• Storage conditions:

  • Store at -20°C for long-term storage

  • Keep at 2-8°C for short-term use (up to 1 week)

  • Maintain in recommended buffer conditions (typically PBS with stabilizers and glycerol)

• Aliquoting strategy:

  • Prepare small single-use aliquots to avoid freeze-thaw cycles

  • Use sterile conditions when preparing aliquots

  • Label with date, concentration, and lot number

• Freeze-thaw management:

  • Minimize freeze-thaw cycles as explicitly recommended

  • Thaw antibodies on ice or at 4°C, never at room temperature

  • Return to storage promptly after use

• Working dilution preparation:

  • Prepare fresh working dilutions for each experiment

  • Use high-quality diluents (filtered, sterile)

  • Include appropriate preservatives for extended use

• Shipping and receiving:

  • Upon receipt, store immediately at recommended temperature

  • Verify cold chain maintenance during shipping

  • Inspect for signs of degradation before use

• Record-keeping:

  • Document performance across different applications and conditions

  • Track lot-to-lot variations

  • Maintain validation data for reference