EARS2 Antibody

What is EARS2 and what cellular functions does it perform?

EARS2 (glutamyl-tRNA synthetase 2, mitochondrial) is a non-discriminating glutamyl-tRNA synthetase that catalyzes aminoacylation of both mitochondrial tRNA(Glu) and tRNA(Gln), playing a critical role in mitochondrial protein translation . The protein functions through a two-step reaction mechanism: first activating glutamate with ATP to form Glu-AMP, then transferring the glutamate to the acceptor end of either tRNA(Glu) or tRNA(Gln) . EARS2 is classified as a class I aminoacyl-tRNA synthetase and is considered part of the "minimal set" of seventeen aminoacyl-tRNA synthetases found in all living organisms, highlighting its evolutionary importance . The protein is primarily localized to mitochondria and is essential for mitochondrial protein synthesis and cellular viability.

What are the available EARS2 antibody types and their specifications?

Current research-grade EARS2 antibodies include rabbit polyclonal antibodies that target specific regions of the human EARS2 protein. For example, antibody ab225913 is generated against a recombinant fragment corresponding to amino acids 1-200 of human EARS2 . Similarly, the Proteintech antibody (17539-1-AP) recognizes both the 58 kDa and 60 kDa isoforms of EARS2 . These antibodies have been validated for immunohistochemistry on paraffin-embedded tissues (IHC-P) and Western blot (WB) applications . When selecting an EARS2 antibody for research, consideration should be given to the specific applications (IHC-P, WB) and species reactivity (human, mouse, rat) required for your experimental design.

How can researchers validate EARS2 antibody specificity for experimental applications?

Validating antibody specificity requires multiple complementary approaches. First, perform Western blot analysis using positive control samples with known EARS2 expression (e.g., human fibroblasts) to confirm detection of bands at the expected molecular weights (58 and 60 kDa) . Second, include negative controls such as EARS2 knockdown cells or tissues from knockout models when available. Third, correlate immunostaining patterns with known EARS2 subcellular localization (mitochondrial). Fourth, perform peptide competition assays where pre-incubation of the antibody with its immunizing peptide should abolish specific staining. Finally, compare results using alternative antibodies targeting different epitopes of EARS2 to confirm consistency in detection patterns. For IHC applications, include tissue-specific positive controls such as human ovarian carcinoma or adrenal gland tissues, which have demonstrated reliable EARS2 staining patterns .

What methodological approaches are most effective for studying EARS2 function in mitochondrial disease models?

A multi-faceted approach yields the most comprehensive insights into EARS2 function in mitochondrial disease models. Oxygen consumption rate (OCR) and maximum respiratory rate (MRR) measurements using platforms like the SeaHorse FX-96 apparatus provide crucial functional data on mitochondrial respiration efficiency . These parameters should be assessed in both naive conditions and after genetic rescue experiments. Western blot quantification using antibodies against EARS2 (with GAPDH as loading control) allows for protein expression level comparison between patient-derived and control samples . For genetic analysis, whole-exome sequencing followed by Sanger validation of variants provides the foundation for genotype-phenotype correlations. Additionally, complementation studies through lentiviral transduction of wild-type EARS2 cDNA into patient fibroblasts can demonstrate functional rescue of mitochondrial defects, confirming the pathogenicity of identified variants . This approach has successfully shown recovery of defective respiratory parameters in EARS2-deficient cell lines.

How should researchers interpret contradictory data between functional EARS2 variants and clinical phenotypes?

Interpretation of contradictory data requires careful consideration of multiple factors. As demonstrated in research by the case with compound heterozygous EARS2 mutations (c.328G>A/p.G110S and c.1045G>A/p.E349K), functionally validated pathogenic variants may not always be clinically relevant to the patient's actual disease . When faced with such contradictions, researchers should:

• Evaluate the full genetic background through comprehensive sequencing to identify potential confounding variants in other genes

• Perform tissue-specific functional studies that relate directly to the affected organs in the clinical phenotype

• Consider the possibility of genetic modifiers that may influence phenotypic expression

• Assess allele-specific effects through RNA sequencing to determine if there is differential expression of wild-type versus mutant alleles

• Compare biochemical phenotypes across multiple patient samples with similar variants

The research literature demonstrates that variants with demonstrated impact on protein function should not necessarily be considered clinically relevant without corroborating evidence from multiple patients with similar phenotypes and genotypes .

What are the current limitations in experimental methods for assessing EARS2 antibody cross-reactivity?

Current experimental methods for assessing EARS2 antibody cross-reactivity face several limitations. First, the high sequence homology between aminoacyl-tRNA synthetase family members creates potential for cross-recognition, which standard Western blotting may not fully resolve. Second, conventional validation using knockout models is challenging due to the essential nature of EARS2 for cellular viability. Third, post-translational modifications of EARS2 in different tissues may alter epitope accessibility, leading to variable antibody performance across sample types. Fourth, the existence of multiple isoforms (58 and 60 kDa) complicates interpretation of band patterns . To address these limitations, researchers should employ competitive binding assays with recombinant EARS2 and related synthetases, use mass spectrometry for definitive protein identification in immunoprecipitated samples, and validate findings across multiple antibodies targeting different EARS2 epitopes. Additionally, careful titration experiments should be performed to determine optimal antibody concentrations that maximize specific signal while minimizing background.

How do EARS2 mutations correlate with specific mitochondrial disease phenotypes?

EARS2 mutations have been primarily associated with leukoencephalopathy with thalamus and brainstem involvement and high lactate (LTBL), an autosomal recessive disorder affecting the white matter of the brain . Research demonstrates that different mutations can lead to varying degrees of protein dysfunction and clinical severity. For example, the c.328G>A (p.G110S) mutation located in exon 3 in the catalytic domain has been found in multiple patients with LTBL . Functional studies on patient fibroblasts carrying compound heterozygous mutations (c.328G>A/p.G110S and c.1045G>A/p.E349K) have shown significant mitochondrial dysfunction with approximately 11% decrease in oxygen consumption rate and 43% decrease in maximum respiratory rate compared to controls . Additionally, these mutations resulted in EARS2 protein levels reduced to 30% of normal controls . The severity of the biochemical defect appears to correlate with clinical presentation, though genetic background and environmental factors may modify the phenotypic expression.

What experimental evidence supports the pathogenicity of specific EARS2 variants?

Multiple lines of experimental evidence support the pathogenicity of specific EARS2 variants. In vitro functional studies provide the strongest evidence, particularly mitochondrial respiration assays measuring oxygen consumption rate (OCR) and maximum respiratory rate (MRR) . For example, fibroblasts harboring the compound heterozygous mutations c.328G>A (p.G110S) and c.1045G>A (p.E349K) showed significant reductions in both parameters . Western blot analysis demonstrated reduced EARS2 protein levels (30% of normal controls) in these cells . Critically, genetic complementation through transduction with wild-type EARS2 cDNA rescued the defective respiratory parameters, confirming the causal relationship between the variants and mitochondrial dysfunction .

Additionally, in silico analyses provide supporting evidence: the G110 residue is evolutionarily invariant from humans through Caenorhabditis elegans, while the E349 residue shows amino acid class conservation . Most pathogenicity prediction programs (SIFT, PolyPhen2, MutationTaster, LRT) classify these variants as damaging or disease-causing . Population frequency data also supports pathogenicity, as these variants occur at very low frequencies in population databases (MAF 0.03-0.18%) .

What methodological approaches should be used to distinguish pathogenic EARS2 variants from non-pathogenic polymorphisms?

Distinguishing pathogenic EARS2 variants from non-pathogenic polymorphisms requires a systematic multi-tiered approach. The following methodology is recommended based on current research practices:

True pathogenic variants typically show consensus across in silico predictions, high evolutionary conservation, low population frequency (<0.1%), reduced protein expression, measurable functional deficits, successful complementation rescue, and recurrence in patients with similar phenotypes . The c.328G>A (p.G110S) variant provides a strong example, having been identified in multiple unrelated patients with LTBL, demonstrating both functional defects and clinical relevance .

How should researchers design experiments to investigate the impact of EARS2 antibody specificity on experimental outcomes?

When investigating EARS2 antibody specificity impacts on experimental outcomes, a systematic approach should include parallel experiments with multiple antibodies targeting different EARS2 epitopes. Begin with a characterization phase using Western blot analysis comparing commercially available antibodies on a panel of cell lines with varying EARS2 expression levels. Implement siRNA knockdown controls to verify signal reduction correlates with EARS2 depletion. For immunohistochemistry applications, conduct side-by-side comparisons of staining patterns in tissues known to express EARS2, such as human ovarian carcinoma and adrenal gland tissues .

To quantify specificity differences, develop a standardized scoring system for background staining and signal-to-noise ratio. Additionally, perform epitope mapping to identify which antibodies target conserved domains that might cross-react with other aminoacyl-tRNA synthetases. For critical applications, validate key findings using orthogonal methods such as RNA expression analysis or mass spectrometry. Finally, establish a laboratory-specific validation protocol that includes positive and negative controls appropriate for each experimental context, recognizing that antibody performance may vary by application (Western blot vs. IHC) and sample preparation method.

What are the optimal experimental conditions for detecting low abundance EARS2 protein in various tissue samples?

Detecting low abundance EARS2 protein requires optimized protocols tailored to specific tissue types. For Western blot analysis, implement an enrichment strategy beginning with subcellular fractionation to isolate mitochondria, where EARS2 is predominantly localized. Enhanced chemiluminescence (ECL) systems with extended exposure times and highly sensitive detection instruments improve signal detection. Consider sample loading of at least 50 μg of protein per lane, as used in published EARS2 studies . For immunohistochemistry, heat-induced epitope retrieval methods significantly improve antigen accessibility in paraffin-embedded tissues. Validated dilutions for EARS2 antibodies in IHC-P applications (e.g., 1/100 for ab225913) provide a starting point for optimization .

Signal amplification techniques such as tyramide signal amplification can increase detection sensitivity by 10-100 fold over conventional methods. For immunofluorescence applications, confocal microscopy with z-stack acquisition improves detection of focal signals. Importantly, all optimization efforts should include appropriate positive controls (tissues known to express EARS2) and negative controls (including peptide competition assays) to distinguish specific signals from background or non-specific binding.

What key controls should be implemented when using EARS2 antibodies for studying disease-associated variants?

Implementing robust controls is essential when using EARS2 antibodies to study disease-associated variants. Primary controls should include:

• Wild-type cell lines/tissues paired with patient-derived samples carrying EARS2 variants, enabling direct comparison of antibody reactivity patterns

• Genetic rescue controls using lentiviral transduction of wild-type EARS2 cDNA into patient fibroblasts, which should restore protein levels and mitochondrial function if the antibody is specifically detecting EARS2

• Loading controls for Western blot (e.g., GAPDH) to ensure equal protein loading and facilitate accurate quantification of EARS2 expression differences

• Isotype controls matched to the EARS2 antibody host species and immunoglobulin class to identify non-specific binding

• Peptide competition assays where pre-incubation of the antibody with immunizing peptide should abolish specific staining

• Parallel assays using antibodies targeting different EARS2 epitopes to confirm consistency in detected expression patterns

• RNA-level expression analysis (RT-qPCR) as an orthogonal method to confirm protein-level findings

These controls collectively ensure that observed differences between wild-type and variant EARS2 reflect actual biological variations rather than technical artifacts or non-specific antibody binding.

How can EARS2 antibodies be utilized for investigating mitochondrial translation defects in neurodegenerative disorders?

EARS2 antibodies provide valuable tools for investigating mitochondrial translation defects in neurodegenerative disorders through multiple research applications. Immunohistochemistry using validated EARS2 antibodies can map the distribution and abundance of EARS2 protein in brain tissue sections from patients with various neurodegenerative conditions compared to age-matched controls . This approach allows correlation between EARS2 levels and pathological features. Co-immunoprecipitation studies using EARS2 antibodies can identify novel protein-protein interactions that may be disrupted in disease states, potentially revealing how EARS2 dysfunction contributes to neurodegeneration pathways.

For functional studies, researchers can combine EARS2 antibodies with respirometry assays to correlate EARS2 protein levels with mitochondrial respiratory chain activity in patient-derived neurons or brain organoids . Single-cell imaging approaches using fluorescently-labeled EARS2 antibodies can track real-time changes in EARS2 localization during cellular stress, providing insights into dynamic responses to neurodegenerative triggers. Additionally, researchers should consider developing proximity ligation assays using EARS2 antibodies to visualize interactions between EARS2 and other mitochondrial translation machinery components in situ, potentially identifying disrupted interactions in disease states.

How might advanced research techniques enhance our understanding of EARS2 biology in mitochondrial disease pathogenesis?

Advanced research techniques offer transformative potential for understanding EARS2 biology in mitochondrial disease pathogenesis. CRISPR-Cas9 gene editing enables creation of isogenic cell lines with specific EARS2 variants, allowing direct comparison of variant effects while controlling for genetic background. These models can be further developed into three-dimensional organoids that better recapitulate tissue-specific disease manifestations. Single-cell multi-omics approaches combining transcriptomics, proteomics, and metabolomics at the individual cell level can reveal cell type-specific responses to EARS2 dysfunction, potentially explaining the neurological tropism of EARS2-related disorders.

Live-cell imaging techniques using EARS2 tagged with fluorescent reporters can track protein localization, interactions, and dynamics in real-time. Super-resolution microscopy approaches can visualize EARS2 within the mitochondrial translation machinery at nanometer resolution, revealing structural insights previously unobtainable. Cryo-electron microscopy offers potential for determining the three-dimensional structure of EARS2 in complex with its tRNA substrates, enhancing understanding of how specific mutations disrupt function. Additionally, patient-derived induced pluripotent stem cells differentiated into affected cell types (e.g., oligodendrocytes, neurons) provide platforms for high-throughput drug screening to identify compounds that rescue EARS2 dysfunction. These advanced techniques collectively promise to bridge current knowledge gaps and accelerate therapeutic development for EARS2-related disorders.

What are the most significant considerations for researchers selecting EARS2 antibodies for specific experimental applications?

When selecting EARS2 antibodies for specific experimental applications, researchers must prioritize several critical considerations. First, application compatibility should be verified through published validation data, as antibodies optimized for Western blot may not perform equivalently in immunohistochemistry or immunoprecipitation . Second, epitope location is crucial—antibodies targeting different domains of EARS2 (e.g., catalytic domain versus anticodon binding domain) may yield different results, particularly when studying truncated variants or specific mutations. Third, species cross-reactivity must be confirmed if working with model organisms, as some antibodies may recognize human EARS2 but not mouse or rat orthologs despite sequence homology .

Fourth, lot-to-lot consistency should be assessed through internal validation protocols, as manufacturing variations can affect performance. Fifth, researchers should consider the ability to detect both EARS2 isoforms (58 and 60 kDa) if studying differential isoform expression . Sixth, sensitivity thresholds must be established for each application to ensure reliable detection of physiological EARS2 levels. Finally, researchers should maintain awareness of potential cross-reactivity with other aminoacyl-tRNA synthetases, implementing appropriate specificity controls. These considerations, integrated into a systematic antibody validation workflow, will maximize experimental reproducibility and reliability when investigating EARS2 biology in health and disease contexts.

How should researchers integrate EARS2 antibody-based methods with other experimental approaches to maximize insights into mitochondrial translation disorders?

Effective research strategies for mitochondrial translation disorders require thoughtful integration of EARS2 antibody-based methods with complementary approaches. A multi-modal framework should begin with genetic analysis (whole-exome/genome sequencing) to identify variants, followed by Sanger validation and in silico pathogenicity prediction . EARS2 antibody-based protein quantification via Western blot provides essential data on expression levels, which should be correlated with functional assays measuring mitochondrial respiration (OCR, MRR) to establish relationships between protein abundance and functional outcomes .

Genetic complementation studies using wild-type EARS2 cDNA should be conducted alongside antibody-based protein detection to confirm restoration of both protein levels and function . Structural biology approaches including cryo-EM can provide insights into how variants affect EARS2 protein conformation. Metabolomic profiling should be integrated to identify downstream metabolic consequences of EARS2 dysfunction. For tissue-specific effects, immunohistochemistry using validated EARS2 antibodies should be combined with tissue-specific functional assessments. Finally, patient-derived cellular models enable longitudinal studies of disease progression and therapeutic responses. This integrated approach leverages the strengths of each method while compensating for individual limitations, providing comprehensive insights into the pathophysiology of EARS2-related mitochondrial translation disorders.

What emerging methodologies might address current limitations in EARS2 research?

Several emerging methodologies show promise for addressing current limitations in EARS2 research. Proximity labeling approaches such as BioID or APEX2 fused to EARS2 can identify transient interacting partners in living cells, revealing the dynamic EARS2 interactome under various physiological and pathological conditions. Single-molecule imaging techniques including single-molecule FRET (Förster Resonance Energy Transfer) can visualize individual EARS2 molecules during the aminoacylation reaction, providing unprecedented insights into the catalytic mechanism and how disease-associated mutations disrupt function.