MTERF4 Antibody

What is MTERF4 and why is it important in mitochondrial research?

MTERF4 (Mitochondrial Transcription Termination Factor 4) is a 44 kDa mitochondrial protein comprised of 381 amino acid residues in humans that plays a critical role in mitochondrial ribosome biogenesis and translation . The importance of MTERF4 stems from its function as a regulator that forms a stoichiometric complex with NSUN4, a ribosomal RNA methyltransferase, facilitating recruitment of this enzyme to the large ribosomal subunit (39S) . Knockout studies have demonstrated that loss of MTERF4 leads to defective ribosomal assembly and drastically reduced mitochondrial translation, making it an essential protein for proper mitochondrial function . Additionally, MTERF4 is conserved across species with orthologs identified in mouse, rat, bovine, frog, zebrafish, chimpanzee, and chicken, highlighting its evolutionary significance in mitochondrial gene expression regulation .

How should researchers select the appropriate MTERF4 antibody for their specific application?

Selection of the appropriate MTERF4 antibody should be guided by:

• Experimental application: Consider whether your application is Western Blot, ELISA, Immunofluorescence, Immunohistochemistry, or Immunocytochemistry. For instance, Western Blot is the most common application for MTERF4 antibodies, with recommended dilutions typically ranging from 1:500 to 1:2000 .

• Species reactivity: Verify that the antibody recognizes MTERF4 in your species of interest. Available antibodies show reactivity with various species including human, mouse, rat, rabbit, bovine, and others .

• Epitope consideration: Some antibodies target specific regions of MTERF4, such as the C-terminal region, which may affect recognition depending on potential isoforms or post-translational modifications .

• Conjugation requirements: Determine whether your experiment requires unconjugated antibodies or those with specific conjugates (HRP, biotin, Alexa Fluor, etc.) based on your detection method .

• Validation evidence: Review whether the antibody has been validated in your specific application through published literature or manufacturer testing data .

What are the molecular characteristics of MTERF4 that researchers should consider when designing experiments?

When designing experiments involving MTERF4, researchers should consider these key molecular characteristics:

• Structural properties: MTERF4 contains MTERF repeats that form a half-donut shaped, right-handed superhelix, with the concave side displaying a positively charged path for nucleic acid interaction .

• Dual molecular forms: The mature mitochondrial MTERF4 protein exists in two forms with different N-termini, one starting at residue 43 and another at residue 48 .

• Observed molecular weight: While calculated at 44 kDa, MTERF4 is often observed at approximately 37 kDa in Western blots, which should be considered when analyzing results .

• Nucleic acid binding capability: MTERF4 binds to mitochondrial ribosomal RNAs (16S, 12S, and 7S), which is essential for its function in ribosome biogenesis .

• Complex formation: MTERF4 forms a functional complex with NSUN4 methyltransferase, and this interaction is critical for proper mitochondrial translation .

What are the optimal conditions for using MTERF4 antibodies in Western blot analysis?

Optimal Western blot conditions for MTERF4 antibodies include:

• Sample preparation:

  • Extract proteins from mitochondrial fractions for enriched detection

  • For subcellular localization studies, prepare separate fractions of leaf/tissue, chloroplast, and mitochondrial extracts as demonstrated in Hammani et al.'s work with Zm-mTERF4

• Antibody dilution:

  • Typically 1:500-1:2000 as recommended for many commercial MTERF4 antibodies

  • Optimize through titration in each specific testing system

• Expected band size:

  • Look for bands at approximately 37 kDa, although the calculated molecular weight is 44 kDa

  • In plant systems like maize, detection is slightly ahead of the 50 kDa marker, consistent with mature Zm-mTERF4 (49 kDa)

• Controls:

  • Include both positive controls (cell lines with known MTERF4 expression such as HT-1080, HeLa, HepG2, or LNCaP cells)

  • Negative controls should ideally include MTERF4 knockdown samples

• Detection method:

  • Enhanced chemiluminescence systems are commonly used

  • For quantitative analysis, consider fluorescence-based detection systems

How can researchers effectively validate MTERF4 antibody specificity for their experiments?

To validate MTERF4 antibody specificity, researchers should employ these methodological approaches:

• Genetic validation:

  • Test antibody against tissues/cells from MTERF4 knockout or knockdown models

  • As demonstrated in the Zm-mTERF4 study, antibody detection should be diminished in MTERF4-deficient samples

• Peptide competition assay:

  • Pre-incubate antibody with the immunogen peptide before application

  • Specific signals should be blocked by this competition

• Multiple antibody concordance:

  • Compare results using antibodies targeting different epitopes of MTERF4

  • Consistent detection patterns increase confidence in specificity

• Recombinant protein controls:

  • Express recombinant MTERF4 (as done for Zm-mTERF4 using the pMAL-TEV system)

  • Use as a positive control for size verification

• Mass spectrometry confirmation:

  • Immunoprecipitate MTERF4 using the antibody

  • Confirm identity of the precipitated protein by mass spectrometry

What methodologies are recommended for analyzing MTERF4's interactions with mitochondrial RNA and protein complexes?

For analyzing MTERF4's interactions with mitochondrial RNA and protein complexes, these methodologies are recommended:

• RNA coimmunoprecipitation (RIP):

  • Use affinity-purified MTERF4 antibodies for immunoprecipitation from stromal extracts

  • Analyze coimmunoprecipitating RNAs by hybridization to tiling microarrays of the mitochondrial or chloroplast genome

  • This approach revealed MTERF4's association with group II introns in chloroplasts

• Protein complex analysis:

  • Size exclusion chromatography to identify high molecular weight complexes containing MTERF4

  • Blue native gel electrophoresis to maintain native protein complexes

  • Co-immunoprecipitation with antibodies against known MTERF4 partners (e.g., NSUN4)

• Cross-linking methodologies:

  • RNA-protein crosslinking followed by immunoprecipitation to identify direct RNA binding sites

  • Protein-protein crosslinking to capture transient interactions

• Subcellular fractionation:

  • Differential centrifugation to isolate mitochondrial, chloroplast, and stromal fractions

  • Further subfractionate mitochondria to determine precise localization

  • Western blotting of fractions can verify MTERF4 localization to mitochondria or chloroplasts

• Mass spectrometry approaches:

  • MS analysis of MTERF4-containing complexes to identify all interacting partners

  • Quantitative proteomics to measure changes in complex composition under different conditions

How do MTERF4 antibodies contribute to understanding mitochondrial translation regulation in disease models?

MTERF4 antibodies have been instrumental in elucidating the role of this protein in mitochondrial translation regulation and disease pathology:

• Parkinson's disease models:

  • Studies using MTERF3 (a related family member) have demonstrated its regulatory role in MPP+-induced cellular models of Parkinson's disease

  • Similar approaches with MTERF4 antibodies can help determine its potential protective role in neurodegenerative conditions

• Embryonic lethality models:

  • Knockout studies of MTERF4 in mice have shown embryonic lethality, indicating its essential role in development

  • Antibodies enable tissue-specific analysis of MTERF4 expression and correlation with developmental defects

• Mitochondrial dysfunction analysis:

  • MTERF4 antibodies allow assessment of protein levels in various mitochondrial dysfunction scenarios

  • Western blotting with MTERF4 antibodies can correlate protein expression with mitochondrial respiratory chain component levels

• Splicing defect investigation:

  • In plant models, MTERF4 antibodies have revealed the protein's role in group II intron splicing

  • Immunoprecipitation experiments have identified specific RNA targets affected by MTERF4 deficiency

• Ribosome biogenesis assessment:

  • MTERF4 antibodies enable tracking of its association with ribosomal subunits

  • This helps understand how defective ribosome assembly contributes to translation deficiencies in disease states

What are the recommended controls when using MTERF4 antibodies to study mitochondrial ribosome biogenesis?

When studying mitochondrial ribosome biogenesis with MTERF4 antibodies, these controls are essential:

How do plant and animal MTERF4 functions differ, and what experimental approaches can distinguish these roles?

Plant and animal MTERF4 exhibit both distinct and shared functions that can be distinguished through various experimental approaches:

• Functional differences and similarities:

  • In animals: MTERF4 primarily regulates mitochondrial ribosomal biogenesis and translation

  • In plants: MTERF4 (BSM/RUG2/mTERF4 in Arabidopsis, Zm-mTERF4 in maize) promotes splicing of group II introns in chloroplasts and may have dual mitochondrial/chloroplast localization

  • Both contribute to organellar gene expression, though through different mechanisms

• Localization studies:

  • Immunoblotting of fractionated organelles using MTERF4 antibodies

  • Zm-mTERF4 localizes to chloroplasts and not mitochondria, while Arabidopsis BSM/RUG2/mTERF4 has been reported in both organelles

  • Animal MTERF4 is exclusively mitochondrial

• RNA association analysis:

  • RNA coimmunoprecipitation followed by sequencing or microarray analysis

  • In plants, MTERF4 associates with chloroplast group II introns

  • In animals, MTERF4 binds mitochondrial ribosomal RNAs

• Protein complex characterization:

  • Co-immunoprecipitation to identify interacting partners

  • Animal MTERF4 interacts with NSUN4

  • Plant MTERF4 forms complexes with chloroplast splicing factors

• Developmental impact comparison:

  • Knockout/knockdown phenotype assessment

  • Both plant and animal MTERF4 mutants show severe developmental defects, with knockout often causing embryonic lethality

What strategies can researchers employ when MTERF4 antibodies show cross-reactivity with other MTERF family proteins?

When facing cross-reactivity issues with MTERF4 antibodies against other MTERF family members, researchers can employ these strategies:

• Epitope-specific antibody design:

  • Generate antibodies against unique regions of MTERF4 that lack homology with other family members

  • Target variable regions outside the conserved MTERF motifs

• Pre-absorption techniques:

  • Incubate antibodies with recombinant proteins of related MTERF family members

  • This depletes cross-reactive antibodies before experimental use

• Knockout/knockdown validation:

  • Test antibodies on samples with specific MTERF4 depletion

  • Differential signal reduction compared to other family members confirms specificity

• Two-dimensional Western blotting:

  • Separate proteins by both isoelectric point and molecular weight

  • This can resolve MTERF4 from other family members with similar molecular weights

• Mass spectrometry verification:

  • Following immunoprecipitation, analyze by mass spectrometry

  • Peptide identification confirms which MTERF proteins are actually being detected

How can researchers optimize MTERF4 immunoprecipitation protocols for studying RNA binding patterns?

To optimize MTERF4 immunoprecipitation for RNA binding studies, researchers should:

• Crosslinking optimization:

  • Test different crosslinking methods (UV, formaldehyde) and durations

  • Find balance between preserving interactions and maintaining antibody recognition

• Antibody selection and coupling:

  • Use affinity-purified antibodies with demonstrated specificity

  • Covalently couple antibodies to solid supports to prevent interference from heavy chains

  • In previous studies, affinity-purified Zm-mTERF4 antibodies successfully immunoprecipitated RNA-protein complexes

• Buffer composition:

  • Optimize salt concentration to maintain specific interactions while reducing background

  • Include RNase inhibitors to prevent degradation

  • Consider detergent types and concentrations that preserve protein-RNA interactions

• Control immunoprecipitations:

  • Perform parallel IPs with non-specific IgG

  • Include MTERF4-depleted samples as negative controls

  • Use known RNA targets as positive controls

• RNA recovery and analysis:

  • Optimize RNA extraction methods from immunoprecipitates

  • Consider downstream analysis methods:

    • RT-qPCR for targeted analysis of specific transcripts

    • RNA-seq or microarray for global analysis

    • Structure-specific methods for analyzing intron binding

What are the best approaches for investigating MTERF4 expression changes in response to mitochondrial stress?

To investigate MTERF4 expression changes under mitochondrial stress conditions, researchers should consider:

• Stress induction protocols:

  • Chemical stressors: CCCP, rotenone, antimycin A, or MPP+ (as used in MTERF3 studies)

  • Metabolic stress: glucose deprivation, hypoxia

  • Genetic stress: mtDNA depletion, respiratory complex inhibition

• Time-course analysis:

  • Collect samples at multiple time points post-stress induction

  • Analyze both acute and chronic stress responses

  • Monitor MTERF4 protein levels by Western blotting with optimized antibody dilutions

• Subcellular fractionation:

  • Isolate mitochondria to directly assess local MTERF4 changes

  • Compare cytosolic versus mitochondrial MTERF4 to detect potential redistribution

• Correlative analyses:

  • Simultaneously measure MTERF4 levels and mitochondrial function parameters

  • Assess mitochondrial membrane potential, ROS production, and ATP synthesis

  • Monitor levels of other mitochondrial proteins, particularly NSUN4 and ribosomal components

• Transcriptional and translational regulation:

  • qPCR for MTERF4 mRNA levels

  • Polysome profiling to assess translational regulation

  • Use of proteasome inhibitors to determine if stress-induced changes involve protein degradation

How might new antibody technologies enhance the study of MTERF4 dynamics in live cells?

Emerging antibody technologies offer promising approaches to study MTERF4 dynamics in live cells:

• Nanobody development:

  • Single-domain antibody fragments against MTERF4 could enable:

    • Intracellular expression for live-cell imaging

    • Reduced interference with MTERF4 function due to smaller size

    • Better penetration into mitochondrial compartments

• FRET-based proximity sensors:

  • Fluorophore-coupled MTERF4 antibodies paired with antibodies against interaction partners

  • Would allow real-time monitoring of complex formation between MTERF4 and NSUN4 or other factors

• Split-GFP complementation systems:

  • Tag MTERF4 with one GFP fragment and potential interactors with complementary fragments

  • Fluorescence indicates interaction in living cells

  • Would provide spatial and temporal resolution of MTERF4 complex formation

• Antibody-based optogenetic tools:

  • Photocaged antibody fragments that can be activated by light

  • Would allow temporal control of MTERF4 inhibition in specific cellular compartments

• Intrabodies with conditional stability domains:

  • Engineered antibody fragments against MTERF4 with regulatable stability

  • Would permit inducible disruption of MTERF4 function in specific subcellular locations

What are the most promising approaches for investigating tissue-specific roles of MTERF4 using antibodies?

For investigating tissue-specific roles of MTERF4, these approaches show the most promise:

• Multi-tissue expression profiling:

  • Systematic immunohistochemistry or immunoblotting across tissues

  • Correlate MTERF4 levels with tissue-specific mitochondrial activity

  • Compare different developmental stages to identify temporal regulation patterns

• Conditional knockout models with antibody validation:

  • Generate tissue-specific MTERF4 knockout models

  • Use antibodies to confirm knockout efficiency

  • Analyze phenotypic consequences in relation to MTERF4 levels

  • This approach could extend findings from embryonic lethal models to adult tissues

• Single-cell analysis:

  • Combine MTERF4 antibody-based detection with single-cell isolation techniques

  • Identify cell type-specific expression patterns within heterogeneous tissues

  • Correlate with mitochondrial functional parameters at single-cell resolution

• 3D tissue imaging:

  • Clear tissue technology combined with MTERF4 immunolabeling

  • Would reveal spatial distribution within intact organs

  • Could identify regional specialization of MTERF4 function

• Patient-derived samples:

  • Apply validated MTERF4 antibodies to patient biopsies

  • Compare expression across disease states affecting mitochondrial function

  • Correlate with clinical parameters to establish disease relevance

How can MTERF4 antibodies contribute to understanding the evolutionary divergence of mitochondrial gene expression regulation?

MTERF4 antibodies can significantly contribute to understanding evolutionary divergence in mitochondrial gene expression regulation through:

• Cross-species comparative analysis:

  • Apply validated antibodies across evolutionary diverse species

  • MTERF4 orthologs have been reported in mouse, rat, bovine, frog, zebrafish, chimpanzee, and chicken

  • Systematic comparison would reveal conservation of expression patterns and subcellular localization

• Functional conservation testing:

  • Immunoprecipitate MTERF4 from diverse species and compare:

    • Interacting proteins (especially NSUN4 homologs)

    • Bound RNA species

    • Post-translational modifications

• Structure-function relationship studies:

  • Use antibodies recognizing specific domains to determine if functional regions are accessible across species

  • Compare binding patterns to nucleic acids and proteins

  • Examine if the half-donut shaped, right-handed superhelix structure is conserved

• Plant-animal comparison studies:

  • Leverage findings from plant systems where MTERF4 functions in chloroplast RNA splicing

  • Compare with animal systems where it regulates mitochondrial ribosome biogenesis

  • Use antibodies to trace lineage-specific adaptations in localization and function

• Ancient conserved functions identification:

  • Use antibodies against the most conserved epitopes of MTERF4

  • Test recognition in primitive eukaryotes and prokaryotic ancestors

  • This could reveal the ancestral functions of MTERF proteins before evolutionary divergence