METTL4 Antibody

What is METTL4 and what cellular functions does it perform?

METTL4 is a methyltransferase enzyme that primarily catalyzes N6-methyladenine (6mA) modifications within DNA. Gene ontology annotations indicate that METTL4 possesses methyltransferase activity and nucleic acid binding properties . Recent studies have established that METTL4 preferentially targets mitochondrial DNA (mtDNA), particularly promoter regions, where it catalyzes 6mA modifications . This methylation activity significantly influences transcription initiation complex assembly and subsequent mitochondrial function.

METTL4 has been observed to localize predominantly within the mitochondria of cardiomyocytes, where 6mA modifications were found to be significantly more abundant in mtDNA compared to nuclear DNA . The enzyme's expression levels vary during development and under pathological conditions, suggesting a dynamic regulatory role in cellular function.

What applications are METTL4 antibodies validated for in research?

METTL4 antibodies have been validated for multiple research applications including:

• Western Blotting (WB): Recommended dilutions range from 1:1000 to 1:10000

• Immunohistochemistry (IHC): Recommended dilutions range from 1:20 to 1:200

• Enzyme-Linked Immunosorbent Assay (ELISA)

Experimental validation has confirmed METTL4 antibody specificity in human tissues (particularly testis) and cell lines including HeLa and MCF7. Positive Western blot detection typically observes a molecular weight of approximately 70kD .

How should researchers optimize METTL4 immunohistochemical staining protocols?

When performing immunohistochemistry with METTL4 antibodies, researchers should:

• Use paraffin-embedded tissue sections with optimal thickness (4-6 μm)

• Perform antigen retrieval (preferably heat-induced epitope retrieval in citrate buffer pH 6.0)

• Block endogenous peroxidase activity with 3% hydrogen peroxide

• Apply primary antibody at appropriate dilution (1:20 to 1:200) and incubate overnight at 4°C

• Validate staining patterns against known positive controls such as human testis tissue

• Include negative controls by omitting primary antibody or using non-specific IgG

Researchers should note that METTL4 antibodies have shown successful staining in human testis tissue with clear visualization using a 10x lens . For dual-staining experiments, consider pairing with mitochondrial markers to confirm the subcellular localization observed in cardiomyocytes .

What storage and handling conditions maintain optimal METTL4 antibody activity?

To maintain METTL4 antibody integrity and performance:

• Store antibodies at -20°C in the formulation provided (typically PBS with 0.02% sodium azide and 50% glycerol, pH 7.3)

• DO NOT ALIQUOT the antibody solution to prevent freeze-thaw cycles that can degrade activity

• Avoid repeated freeze-thaw cycles; thaw only once before use

• When working with the antibody, keep on ice and return to -20°C immediately after use

• Observe expiration dates and evaluate antibody performance if stored for extended periods

Proper handling practices significantly impact experimental reproducibility when using METTL4 antibodies for critical applications like cancer biomarker assessment.

How can METTL4 antibodies be employed to study its role as a prognostic biomarker in hepatocellular carcinoma?

Recent research has identified METTL4 as a potential prognostic biomarker in hepatocellular carcinoma (HCC). To effectively investigate this relationship, researchers should:

• Implement a tiered experimental approach:

  • Begin with bioinformatics analysis using databases like TCGA to assess METTL4 expression patterns

  • Validate findings using IHC on patient tissue microarrays with proper controls

  • Correlate expression levels with patient clinicopathological features and outcomes

• Develop staining scoring systems:

  • Use a combined score accounting for both staining intensity and percentage of positive cells

  • Establish appropriate cutoffs for "high" versus "low" expression groups

• Correlate with survival data:

  • Track recurrence-free survival (RFS) as a primary endpoint

  • Perform multivariate Cox regression analysis to determine independent prognostic value

Research has demonstrated that high METTL4 expression significantly correlates with shorter recurrence-free survival in HCC patients. When combined with METTL5 expression and tumor diameter, these factors can stratify patients into distinct risk groups with 3-year RFS rates of 18.75%, 69.70%, and 93.75% for high, medium, and low-risk groups, respectively .

What methodological approaches can detect METTL4-mediated 6mA modifications in mitochondrial DNA?

To detect and quantify METTL4-mediated 6mA modifications in mitochondrial DNA, researchers should consider these validated methodological approaches:

• Methylated DNA immunoprecipitation sequencing (MeDIP-Seq):

  • This technique has successfully characterized 6mA distribution across cardiomyocyte mtDNA

  • The approach revealed a consensus sequence (TGGATC) as a potential preferential site for 6mA modifications

  • Allows for genome-wide analysis of 6mA distribution patterns

• Chromatin immunoprecipitation (ChIP) assay:

  • Effective for validating direct interaction between METTL4 protein and mtDNA promoter regions

  • Can quantify changes in METTL4 occupancy under different experimental conditions

  • Useful for studying competition between METTL4 and transcription factors at promoter regions

• Quantitative comparison between mtDNA and nuclear DNA:

  • Studies have shown 6mA levels in mtDNA are approximately 1400-fold higher than in nuclear DNA

  • Important control to establish specificity of METTL4 targeting

When designing these experiments, researchers should include appropriate controls, such as METTL4 mutants lacking methyltransferase activity (e.g., D286A/W289A mutations) .

How does METTL4 overexpression impact mitochondrial function in experimental models?

Experimental evidence demonstrates that METTL4 overexpression significantly disrupts mitochondrial function through several mechanisms:

• Transcriptional suppression:

  • METTL4 overexpression decreases protein levels of mitochondrial electron transport chain (ETC) complexes

  • Reduces mRNA expression of ETC genes originating from mtDNA transcription

• Respiratory capacity:

  • Compromises both Complex I-supported and Complex II-supported oxygen consumption rate (OCR)

  • Leads to compensatory glycolysis enhancement in affected cells

• Transcription complex interference:

  • METTL4 interferes with the assembly of the transcription initiation complex at mtDNA promoters

  • Specifically obstructs the occupancy of TFAM and POLRMT at mtDNA promoters

  • This occurs both under basal conditions and during TFAM overexpression

Importantly, enzyme-inactive METTL4 mutants (D286A/W289A) do not reproduce these effects, confirming that the methyltransferase activity is essential for METTL4's impact on mitochondrial function .

What strategies can distinguish between specific and non-specific METTL4 antibody binding in complex tissue samples?

Ensuring METTL4 antibody specificity is crucial for accurate interpretation of experimental results. Recommended validation strategies include:

• Multiple antibody validation:

  • Use antibodies targeting different epitopes of METTL4

  • Compare staining patterns between monoclonal and polyclonal antibodies

  • Confirm consistency of results across antibodies from different vendors

• Genetic manipulation controls:

  • Compare staining in wild-type versus METTL4 knockout/knockdown models

  • Use overexpression systems as positive controls

  • Consider doxycycline-inducible systems for titratable expression

• Peptide competition assays:

  • Pre-incubate antibody with excess purified METTL4 recombinant protein

  • Observe elimination of specific signal while non-specific binding persists

  • Include control peptides from unrelated proteins

• Cross-reactivity assessment:

  • Test antibody against related methyltransferases (particularly METTL5)

  • Perform immunoprecipitation followed by mass spectrometry to identify all bound proteins

  • Conduct Western blot against lysates from cells expressing related proteins

For complex tissue samples like liver biopsies used in HCC studies, consider dual immunofluorescence with known markers of subcellular compartments to confirm expected localization patterns, particularly mitochondrial colocalization .

How can researchers investigate the relationship between METTL4 expression and cardiac pathophysiology?

To investigate METTL4's role in cardiac pathophysiology, researchers should consider these methodological approaches:

• Targeted gene delivery systems:

  • Adeno-associated virus serotype 9 (AAV9) vectors carrying the METTL4 gene under cardiac-specific promoters (e.g., cardiac troponin T promoter)

  • Achieve cardiomyocyte-specific expression via intravenous injection

  • Monitor expression by confirming increases in both mRNA and protein levels in left ventricular tissue

• Cell-specific isolation techniques:

  • Separately analyze cardiomyocyte and non-cardiomyocyte populations

  • Confirm targeted expression is limited to cardiomyocytes

  • Compare mtDNA 6mA levels between populations

• Functional assessments:

  • Evaluate mitochondrial function through oxygen consumption rate measurements

  • Assess electron transport chain complex activity

  • Monitor cardiac function parameters through echocardiography

• Molecular mechanism investigation:

  • Track changes in mtDNA promoter methylation through development and disease progression

  • Analyze transcription factor displacement from promoters using ChIP assays

  • Study compensatory metabolic adaptations like enhanced glycolysis

Research has shown that METTL4 expression decreases during postnatal cardiomyocyte maturation but increases in failing cardiomyocytes, suggesting a shift toward a neonatal-like state in heart failure. Cardiomyocyte-specific deletion of the METTL4 gene has been shown to eliminate mtDNA 6mA excess, preserve mitochondrial function, and mitigate heart failure development in experimental models .

What are common pitfalls when using METTL4 antibodies for IHC, and how can they be overcome?

Common challenges with METTL4 immunohistochemistry and their solutions include:

• High background staining:

  • Increase blocking time and concentration (5% BSA or 10% normal serum)

  • Optimize antibody dilution (start at 1:100 and titrate as needed)

  • Reduce incubation time or temperature for secondary antibody

  • Ensure tissue samples are properly fixed (overfixation can increase background)

• Weak or absent signal:

  • Optimize antigen retrieval (citrate buffer, pH 6.0, 20 minutes)

  • Increase antibody concentration incrementally

  • Extend primary antibody incubation (overnight at 4°C)

  • Switch detection systems to more sensitive methods (e.g., polymer-based detection)

• Inconsistent staining between samples:

  • Standardize fixation time for all specimens

  • Process all samples simultaneously when possible

  • Include positive control tissue (human testis) in each batch

  • Implement automated staining platforms for consistency

• Non-specific nuclear staining:

  • Include additional blocking steps with avidin/biotin when using biotin-based detection

  • Switch to polymer-based detection systems

  • Reduce primary antibody concentration

  • Add 0.1-0.3% Triton X-100 to washing buffers

METTL4 antibodies have been successfully used at 1:100 dilution for IHC in human testis tissue, which serves as an excellent positive control .

How can researchers successfully integrate METTL4 analysis in multi-parameter experimental designs?

For complex experimental designs incorporating METTL4 analysis alongside other parameters:

• Sequential immunostaining approaches:

  • Begin with METTL4 staining using conventional IHC

  • Follow with sequential staining for additional markers

  • Consider multiplex IHC systems with tyramide signal amplification

  • Use spectral imaging systems to resolve overlapping signals

• Correlation with functional outcomes:

  • Design experimental workflows that link METTL4 expression to:

    • Mitochondrial function parameters (OCR, ATP production)

    • Cell cycle and proliferation markers

    • Apoptosis indicators

    • Disease-specific endpoints (e.g., fibrosis, inflammation)

• Integration with -omics datasets:

  • Correlate METTL4 expression with transcriptomic profiles

  • Investigate relationships with metabolomic signatures

  • Explore connections to epigenomic patterns beyond 6mA

  • Develop integrated computational models

Researchers studying hepatocellular carcinoma should consider integrating METTL4 expression analysis with established prognostic factors like maximum tumor diameter to develop comprehensive risk stratification models, as demonstrated in recent studies achieving 3-year RFS rates predictions with high accuracy .