MTERF2 Antibody

What is MTERF2 and why is it important to study?

MTERF2 (also known as MTERFD3) is a member of the mitochondrial transcription termination factors (MTERFs) family that plays crucial roles in regulating mitochondrial gene expression. In mammals, MTERF2 modulates mitochondrial DNA transcription and affects oxidative phosphorylation (OXPHOS) system functionality . Research has demonstrated that inactivation of MTERF2 in mice results in myopathy and memory deficits associated with decreased levels of mitochondrial transcripts and imbalanced tRNA pools, leading to reduced steady-state levels of OXPHOS proteins and diminished respiratory function . MTERF2 binds to the mtDNA promoter region, suggesting its involvement in transcription initiation rather than termination . In plants like Arabidopsis, MTERF2 is chloroplast-localized and required for the splicing of specific group IIB introns, with complete loss of function resulting in embryo lethality . Studying MTERF2 is critical for understanding fundamental aspects of mitochondrial biogenesis, energy metabolism, and associated pathologies.

What types of MTERF2 antibodies are currently available for research?

Based on the available literature, researchers have access to several types of MTERF2 antibodies:

• Mouse monoclonal antibodies against human MTERF2: Eight stable positive monoclonal cell lines have been established, with antibody light chains being kappa and heavy chains displaying three subtypes (IgG1, IgG2a, and IgG2b) . These antibodies have been validated for immunoblotting, immunoprecipitation, and immunofluorescence analyses .

• Rabbit polyclonal antibodies: These have been validated for Western blot applications and show reactivity with human, mouse, and rat samples . Western blot validation has been demonstrated across various cell lines .

• Custom antibodies: Some research groups have developed polyclonal antibodies against recombinant MTERF2 for specific applications such as submitochondrial localization studies .

When selecting an antibody, researchers should consider the specific application, species reactivity, and validation status in relation to their experimental design.

How can I validate the specificity of MTERF2 antibodies in my experimental system?

Validating MTERF2 antibody specificity is crucial for obtaining reliable research results. Consider implementing the following methodological approaches:

• Positive and negative control samples: Use tissues or cell lines with known MTERF2 expression levels. The literature indicates that MTERF2 is ubiquitously expressed but shows relatively high levels in heart, brain, kidney, and testis - tissues with high energy demands .

• Knockdown or knockout validation: Compare antibody reactivity in wild-type samples versus those with reduced or absent MTERF2 expression. Techniques might include siRNA knockdown or CRISPR-Cas9 gene editing.

• Recombinant protein controls: Use purified recombinant MTERF2 protein as a positive control. Studies have successfully expressed and purified full-length His-tagged MTERF2 protein (1-385 aa) in E. coli .

• Multiple antibody approach: Compare results using different antibodies targeting distinct epitopes of MTERF2.

• Peptide competition assay: Pre-incubate the antibody with excess purified antigen before immunodetection to verify that signal disappearance occurs with specific binding.

• Cross-reactivity assessment: Test the antibody against related MTERF family members to ensure specificity, particularly MTERF1, MTERF3, and MTERF4, which share structural similarities.

How can MTERF2 antibodies be used to investigate mitochondrial transcription regulation?

MTERF2 antibodies provide powerful tools for investigating mitochondrial transcription regulation through several advanced methodological approaches:

• Chromatin Immunoprecipitation (ChIP): MTERF2 antibodies can be used to identify MTERF2 binding sites on mtDNA. Research has shown that MTERF2 binds to the mtDNA promoter region, suggesting its role in transcription initiation . ChIP experiments followed by sequencing (ChIP-seq) or qPCR (ChIP-qPCR) can map precise binding locations and patterns under different physiological conditions.

• Co-immunoprecipitation (Co-IP): MTERF2 antibodies enable investigation of protein-protein interactions between MTERF2 and other components of the mitochondrial transcription machinery. This approach can help elucidate regulatory complexes and potential post-translational modifications required for MTERF2 function.

• RNA Immunoprecipitation (RIP): This technique can determine if MTERF2 directly interacts with RNA transcripts, similar to methods used for other MTERF family members in studies of RNA processing .

• Proximity Ligation Assays (PLA): Combining MTERF2 antibodies with antibodies against other proteins can visualize protein interactions in situ, providing spatial information about transcriptional complexes within mitochondria.

• Super-resolution microscopy: MTERF2 antibodies coupled with techniques like STORM or PALM can reveal the sub-mitochondrial distribution of MTERF2 in relation to nucleoids and transcription complexes.

These approaches enable detailed investigation of how MTERF2 contributes to the regulation of mitochondrial gene expression under different physiological and pathological conditions.

What is the relationship between MTERF2 function and oxidative phosphorylation, and how can antibodies help investigate this?

MTERF2 plays a critical role in regulating oxidative phosphorylation (OXPHOS) through its effects on mitochondrial gene expression. Research has established that MTERF2 inactivation in mice results in decreased levels of mitochondrial transcripts and imbalanced tRNA pools, leading to reduced steady-state levels of OXPHOS proteins and diminished respiratory function . MTERF2 antibodies can help investigate this relationship through several methodological approaches:

• Protein expression analysis: Western blotting with MTERF2 antibodies alongside antibodies against OXPHOS components (e.g., PsbD, PsaA, Atpβ) can correlate MTERF2 levels with respiratory chain complex expression .

• Immunofluorescence co-localization: MTERF2 antibodies can be used in co-localization studies with OXPHOS components to investigate spatial relationships within mitochondria.

• Blue Native PAGE combined with immunoblotting: This technique can examine how MTERF2 affects the assembly and stability of OXPHOS complexes by comparing wild-type samples with those having altered MTERF2 expression.

• Time-course studies: Using MTERF2 antibodies in time-resolved experiments following induction or repression of MTERF2 can elucidate the temporal relationship between MTERF2 levels and OXPHOS functionality.

• Cell fractionation combined with immunoblotting: This approach can determine how MTERF2 distribution in mitochondrial compartments correlates with OXPHOS function, building on previous submitochondrial fractionation studies showing MTERF2 localization primarily in the matrix .

These methodologies allow researchers to establish mechanistic links between MTERF2 function and the regulation of oxidative phosphorylation, potentially revealing therapeutic targets for mitochondrial disorders.

How do different MTERF2 antibody preparations compare in detecting oligomeric states of the protein?

Research indicates that MTERF2 can form oligomers, likely dimers, which may be functionally significant . Different antibody preparations may vary in their ability to detect these oligomeric states, and researchers should consider the following methodological aspects:

• Native vs. denaturing conditions: When investigating oligomeric states, antibodies should be tested under both native conditions (e.g., Blue Native PAGE, gel filtration) and denaturing conditions (SDS-PAGE). Research has shown that MTERF2 can be identified in an 80 kDa fraction (suggesting dimerization of the 39 kDa protein) under certain extraction conditions (0.5% digitonin, 0.2M KCl) .

• Epitope accessibility considerations: Oligomerization may mask certain epitopes. Antibodies targeting different regions of MTERF2 may show differential sensitivity to various oligomeric forms.

• Cross-linking approaches: Using MTERF2 antibodies after chemical cross-linking can stabilize transient interactions and improve detection of oligomeric states.

• Extraction conditions: The oligomeric state of MTERF2 is sensitive to extraction conditions. Studies have shown that using high salt conditions (0.7M KCl) shifts MTERF2 from an 80 kDa fraction to approximately 40 kDa (monomeric form) .

• Recombinant protein controls: Including purified recombinant MTERF2 subjected to conditions promoting monomeric or dimeric states provides important reference points for interpreting experimental results.

This comparative approach allows researchers to better understand the physiological relevance of MTERF2 oligomerization and its potential regulatory mechanisms.

What are the optimal conditions for using MTERF2 antibodies in Western blotting?

Successful Western blotting with MTERF2 antibodies requires optimization of several parameters based on published protocols and established methodologies:

• Sample preparation: Total protein extraction from tissues or cells should be performed using buffers containing appropriate protease inhibitors. For mitochondrial enrichment, differential centrifugation techniques have been successfully employed .

• Protein loading: Reports indicate successful detection with 25μg protein per lane . For tissues with lower MTERF2 expression, higher protein loading may be necessary.

• Gel percentage: 10-12% SDS-polyacrylamide gels have been effective for separating MTERF2 (predicted molecular weight of 39 kDa) .

• Transfer conditions: Semi-dry transfer systems (e.g., Turbo transfer system, Bio-Rad) with PVDF membranes have been successfully used .

• Blocking conditions: 3% non-fat dry milk in TBST has been reported as effective for reducing background .

• Antibody dilutions: Effective dilutions vary by antibody preparation:

  • Rabbit polyclonal antibodies: 1:1,000 dilution has been reported

  • Secondary antibody (anti-rabbit IgG-HRP): 1:10,000 dilution has been effective

• Detection system: ECL detection systems with exposure times of approximately 180 seconds have provided clear signals .

• Controls: Include positive controls (tissues with known high MTERF2 expression like heart, brain, kidney, or testis) and negative controls (samples with MTERF2 knockdown).

• Expected results: MTERF2 should appear as a single band at approximately 39 kDa under denaturing conditions. Under native conditions, higher molecular weight bands (~80-90 kDa) may indicate dimeric forms .

These optimized conditions should be adjusted based on specific antibody characteristics and experimental requirements.

What are the best practices for immunoprecipitation using MTERF2 antibodies?

Immunoprecipitation (IP) with MTERF2 antibodies requires careful consideration of experimental conditions to maintain protein interactions while ensuring specificity. Based on available research, consider the following methodological approach:

• Lysis buffer selection: For preserving protein-protein interactions, use mild non-ionic detergents such as 0.5% digitonin with moderate salt concentrations (0.2M KCl) . For disrupting interactions, higher salt conditions (0.7M KCl) have been effective .

• Pre-clearing step: To reduce non-specific binding, pre-clear lysates with appropriate control beads or pre-immune serum.

• Antibody immobilization: Options include:

  • Direct coupling to protein A/G beads

  • Pre-immobilization on magnetic beads

  • Using antibody-conjugated agarose beads

• IP conditions: Incubate overnight at 4°C with gentle rotation to maximize specific binding while minimizing non-specific interactions.

• Washing stringency: Balance between removing non-specific binding while preserving specific interactions. A series of washes with decreasing salt concentrations can be effective.

• Elution methods: Consider:

  • Denaturing elution with SDS sample buffer for downstream SDS-PAGE

  • Native elution with excess peptide antigen for functional studies

  • Acidic glycine buffer for antibody-antigen dissociation while preserving other interactions

• Controls: Critical controls include:

  • IgG control from same species as MTERF2 antibody

  • Input samples (pre-IP lysate)

  • Samples from MTERF2 knockdown systems

• Validation approaches:

  • Reciprocal IP with interacting partners

  • Mass spectrometry identification of co-precipitated proteins

  • Western blot confirmation of expected interactions

These approaches enable investigation of MTERF2's protein-protein interactions, post-translational modifications, and complex formation under various physiological conditions.

How can MTERF2 antibodies be effectively used in immunocytochemistry and immunofluorescence?

Immunocytochemistry and immunofluorescence techniques with MTERF2 antibodies require specific optimization to ensure accurate detection of this mitochondrial protein. Based on published methodologies, consider the following approach:

• Sample preparation:

  • Cell fixation: 4% paraformaldehyde (10-15 minutes at room temperature) preserves mitochondrial morphology

  • Permeabilization: 0.2% Triton X-100 (5-10 minutes) allows antibody access to matrix proteins like MTERF2

  • Blocking: 5-10% normal serum (from the species of secondary antibody) reduces background

• Antibody incubation:

  • Primary antibody: Dilutions need optimization; successful visualization has been achieved with tagged MTERF2 constructs

  • Secondary antibody: Fluorophore selection should avoid spectral overlap with mitochondrial markers

• Mitochondrial co-localization markers:

  • MitoTracker dyes: Effective for co-localization studies with MTERF2

  • Antibodies against established mitochondrial proteins (e.g., HSP60, COXIV)

  • For nucleoid co-localization, RAP-RFP has been used as a marker

• Imaging considerations:

  • Confocal microscopy: Necessary to resolve submitochondrial localization

  • Fluorescence collection parameters: 500-550 nm for GFP-tagged constructs and 670-750 nm for chlorophyll/mitochondrial autofluorescence have been effective

  • Z-stack acquisition: Important for accurate co-localization analysis

• Controls and validation:

  • Mitochondrial markers as positive controls for localization

  • MTERF2 knockdown/knockout cells as negative controls

  • Peptide competition controls to confirm antibody specificity

• Expected results:

  • MTERF2 should show punctate staining that co-localizes with mitochondrial markers

  • In mammalian cells, staining pattern should be consistent with matrix localization

  • In plant cells (if examining MTERF2 homologs), chloroplast localization would be expected

These methodological considerations enable accurate visualization of MTERF2's subcellular localization and potential co-localization with other proteins or mtDNA.

How should researchers interpret conflicting results between different MTERF2 antibodies?

When faced with conflicting results using different MTERF2 antibodies, researchers should implement a systematic analytical approach to determine the source of discrepancies and identify the most reliable data:

• Epitope mapping analysis:

  • Determine the precise epitopes recognized by each antibody

  • Assess whether post-translational modifications might affect epitope accessibility

  • Consider whether alternatively spliced MTERF2 variants might explain differential detection

• Validation hierarchy establishment:

  • Prioritize results from antibodies with the most extensive validation (e.g., those validated against knockout/knockdown controls)

  • Consider whether monoclonal antibodies (more specific but detecting single epitopes) or polyclonal antibodies (recognizing multiple epitopes) are more appropriate for the specific application

• Cross-validation with non-antibody methods:

  • Correlate antibody-based results with mRNA expression data

  • Use tagged MTERF2 constructs to verify localization or interaction patterns

  • Employ mass spectrometry to confirm protein identity and modifications

• Experimental condition comparison:

  • Assess whether discrepancies arise from differences in sample preparation (e.g., fixation methods, extraction buffers)

  • Evaluate if oligomeric state detection varies between antibodies under different conditions

  • Compare native versus denaturing conditions to identify conformation-specific detection

• Specificity re-evaluation:

  • Perform side-by-side testing with multiple antibodies on the same samples

  • Include appropriate positive and negative controls for each antibody

  • Consider cross-reactivity with other MTERF family members

• Quantification method standardization:

  • Ensure consistent quantification methods across experiments

  • Use multiple reference proteins for normalization

  • Apply statistical analyses to determine significance of observed differences

Through this systematic approach, researchers can resolve apparent conflicts and develop a more nuanced understanding of MTERF2 biology that accounts for technical variability versus true biological phenomena.

What are common pitfalls in MTERF2 antibody-based experiments and how can they be avoided?

Researchers working with MTERF2 antibodies should be aware of several common pitfalls that can compromise experimental results. Based on the literature and standard immunological techniques, these challenges and their solutions include:

• Non-specific binding:

  • Pitfall: High background or false-positive signals, particularly in Western blots

  • Solution: Optimize blocking conditions (3% non-fat dry milk in TBST has proven effective) ; increase washing stringency; validate antibody specificity against MTERF2-deficient samples

• Epitope masking in oligomeric forms:

  • Pitfall: Inconsistent detection of MTERF2 oligomers, which can exist as dimers under certain conditions

  • Solution: Use multiple antibodies targeting different epitopes; compare native versus denaturing conditions; optimize extraction conditions (consider that 0.7M KCl shifts MTERF2 from dimeric to monomeric forms)

• Mitochondrial preparation artifacts:

  • Pitfall: Loss of mitochondrial integrity affecting MTERF2 localization assessment

  • Solution: Use established protocols for mitochondrial isolation; include markers for different mitochondrial compartments (e.g., HSP60 for matrix, COXIV for inner membrane) ; perform parallel experiments with in situ approaches

• Fixation-dependent epitope accessibility:

  • Pitfall: Variable immunofluorescence results depending on fixation method

  • Solution: Compare multiple fixation protocols; use epitope retrieval methods if necessary; validate with tagged MTERF2 constructs as controls

• Cross-reactivity with other MTERF family members:

  • Pitfall: Antibodies potentially recognizing related proteins (MTERF1, MTERF3, MTERF4)

  • Solution: Validate specificity using recombinant proteins of each family member; include cross-reactivity controls in experimental design

• Species-specific variation:

  • Pitfall: Antibodies may not perform equally across species despite sequence conservation

  • Solution: Validate antibodies specifically for target species; check epitope conservation across species; include appropriate positive controls

• Quantification challenges:

  • Pitfall: Variability in MTERF2 quantification, particularly when comparing across tissues

  • Solution: Use standardized loading controls; apply digital image analysis tools; establish linear detection ranges for each antibody

By anticipating these common pitfalls and implementing appropriate methodological solutions, researchers can significantly improve the reliability and reproducibility of MTERF2 antibody-based experiments.

How can researchers optimize detection of MTERF2 in tissues with low expression levels?

Detecting MTERF2 in tissues with low expression presents significant technical challenges. Based on the literature and standard immunological practices, researchers can employ several optimization strategies:

• Sample enrichment techniques:

  • Mitochondrial isolation: Concentrate MTERF2 by isolating mitochondria before analysis, using established differential centrifugation protocols

  • Subcellular fractionation: Further enrich matrix proteins, where MTERF2 is predominantly localized

  • Immunoprecipitation: Use MTERF2 antibodies to concentrate the protein before detection

• Signal amplification methods:

  • Tyramide signal amplification (TSA): Enhances sensitivity for immunohistochemistry/immunofluorescence

  • Enhanced chemiluminescence (ECL): Use high-sensitivity ECL systems with extended exposure times (up to 180s has been effective)

  • Biotin-streptavidin systems: Provides multilayer amplification for detection

• Detection system optimization:

  • Antibody concentration: Use optimized concentrations of primary antibodies (1:1,000 dilution has been reported effective)

  • Incubation conditions: Extended incubation times (overnight at 4°C) can improve detection of low-abundance proteins

  • Secondary antibody selection: High-affinity, highly cross-adsorbed secondary antibodies minimize background

• Sample loading considerations:

  • Increased protein loading: Load higher amounts of protein (>25μg per lane) for Western blot applications

  • Gradient gels: Improve separation and concentration of target protein bands

  • Loading controls: Use appropriate loading controls specific to mitochondrial proteins

• Tissue-specific considerations:

  • Focus on tissues with higher expression: MTERF2 shows relatively high expression in heart, brain, kidney, and testis

  • Developmental timing: Consider potential temporal variation in expression

  • Stress conditions: Examine tissues under conditions that might upregulate MTERF2 expression

• Technical modifications:

  • Western blot membrane selection: PVDF membranes may provide better protein retention than nitrocellulose for low-abundance proteins

  • Film vs. digital detection: Digital imaging systems with adjustable sensitivity settings may better detect faint signals

  • Blocking optimization: Test different blocking agents to maximize signal-to-noise ratio

Through systematic optimization of these parameters, researchers can significantly improve detection sensitivity for MTERF2 in tissues with naturally low expression levels.

What approaches can resolve contradictions between MTERF2 localization data from different experimental methods?

Contradictions in MTERF2 localization data from different experimental methods require systematic resolution approaches to determine the most accurate biological picture. Based on research methodologies, consider the following strategies:

• Method-specific artifact assessment:

  • Cell fractionation artifacts: Evaluate whether protein redistribution occurs during isolation procedures; compare results from different fractionation protocols

  • Fixation artifacts: Test multiple fixation methods to assess potential epitope masking or protein extraction during preparation

  • Tag-induced mislocalization: Compare native protein localization with tagged constructs of different sizes/positions

• Complementary method integration:

  • Orthogonal approaches: Combine biochemical fractionation data with in situ visualization methods

  • Super-resolution microscopy: Resolve submitochondrial compartments beyond conventional microscopy limitations

  • Proximity labeling techniques: Use APEX2 or BioID fused to MTERF2 to map spatial relationships independently of antibody detection

• Dynamic localization investigation:

  • Time-course experiments: Assess whether MTERF2 shows temporal variation in localization

  • Stress response: Examine localization under conditions that affect mitochondrial function

  • Cell-cycle dependency: Determine if localization varies throughout the cell cycle

• Quantitative co-localization analysis:

  • Digital co-localization metrics: Apply Pearson's or Mander's coefficients to quantify overlap with known markers

  • Distance mapping: Measure spatial relationships between MTERF2 and established markers (e.g., HSP60 for matrix, COXIV for inner membrane)

  • 3D reconstruction: Use Z-stack imaging to assess volumetric co-localization patterns

• Species and tissue-specific variation consideration:

  • Cross-species comparison: Assess whether localization varies between organisms (e.g., human vs. mouse)

  • Tissue-specific patterns: Compare localization across tissues with different metabolic demands

  • Cell-type heterogeneity: Examine potential differences between cell types within tissues

By systematically addressing these considerations, researchers can resolve contradictions and develop a more accurate understanding of MTERF2's true subcellular localization.