NARS2 Antibody

What is NARS2 and what cellular functions does it perform?

NARS2 (asparaginyl-tRNA synthetase 2) is a mitochondrial enzyme that catalyzes the attachment of asparagine amino acids to mitochondrial tRNA Asn. This function is critical for efficient mitochondrial protein synthesis . NARS2 is a housekeeping protein encoded by the nuclear genome but functions within mitochondria, where it plays an essential role in the translation of mitochondrial DNA-encoded proteins necessary for oxidative phosphorylation . The protein has a calculated molecular weight of approximately 54 kDa, though the observed molecular weight in experiments typically ranges from 50-54 kDa .

What disease associations have been established for NARS2 mutations?

Biallelic variants in NARS2 have been associated with combined oxidative phosphorylation deficiency 24 (COXPD24) and autosomal recessive deafness-94 . Clinical manifestations include early-onset generalized epilepsy, myoclonic seizures, severe bilateral hearing impairment, and cardiac and hepatic involvement . More severe cases present with growth retardation, intractable epilepsy, and hearing loss in early infancy, potentially progressing to spastic paraplegia, neurodegeneration, and even death . Despite these significant clinical implications, fewer than 30 NARS2 variants have been reported in the literature, making it a relatively understudied gene with important pathophysiological implications .

What are the optimal tissue types for NARS2 antibody validation?

For western blot applications, NARS2 antibodies have been successfully validated in multiple tissue types including HepG2 cells, human liver tissue, human brain tissue, K-562 cells, and mouse kidney tissue . For immunohistochemistry, positive staining has been demonstrated in human kidney, heart, lung, placenta, and spleen tissues . When designing experiments to validate new NARS2 antibodies, these tissue types should be considered as positive controls. For immunofluorescence studies, HepG2 cells have shown reliable positive detection .

What protocols are recommended for subcellular localization studies of NARS2?

For mitochondrial localization studies of NARS2, the following protocol has proven effective: Culture cells (such as HEK293T) on coverslips and transfect with FLAG-tagged NARS2 constructs. At 24 hours post-transfection, stain cells with 500 nM MitoTracker Deep Red FM (or similar mitochondrial marker) for 30 minutes. Fix cells in 4% paraformaldehyde, permeabilize with 0.25% Triton X-100 in PBS, and block with 1% BSA. For immunostaining, use mouse anti-FLAG monoclonal antibody followed by appropriate fluorophore-conjugated secondary antibody (e.g., Alexa Fluor 555) . Mount coverslips with DAPI-containing medium and analyze using fluorescence microscopy. This approach allows for clear visualization of whether wild-type and mutant NARS2 proteins properly localize to mitochondria.

How should researchers design experiments to investigate NARS2 dimerization?

To study NARS2 dimerization, co-immunoprecipitation experiments have proven effective. Transfect cells (such as HEK293T) with differentially tagged NARS2 constructs (e.g., FLAG-tagged and HA-tagged). After approximately 36 hours, lyse cells and immunoprecipitate using antibodies against one tag (e.g., anti-HA magnetic beads). Analyze immune complexes by SDS-PAGE and western blotting with antibodies against both tags . This approach allows researchers to determine whether NARS2 variants retain dimerization capacity despite mutations. For thorough validation, replicate experiments at least three times and include appropriate controls such as single-tagged constructs and irrelevant protein controls.

What are the key considerations when studying the functional impact of NARS2 variants?

When investigating NARS2 variants, consider multiple functional aspects: (1) Expression levels: Determine if variants affect protein expression through western blotting; (2) Subcellular localization: Assess whether variants properly localize to mitochondria using immunofluorescence co-localization studies; (3) Dimerization capacity: Evaluate the ability of variant proteins to form dimers through co-immunoprecipitation; (4) Enzymatic activity: Measure aminoacylation activity using appropriate biochemical assays . Additionally, for variants affecting splicing (particularly those in non-coding regions), conduct minigene experiments to verify the impact on mRNA processing . For variants affecting protein structure, molecular dynamics studies can reveal changes in binding free energy and protein stability .

How can researchers validate the specificity of NARS2 antibodies?

To validate NARS2 antibody specificity, implement multiple approaches: (1) Positive and negative control tissues (human liver and brain tissues typically express NARS2, while certain cell lines may serve as negative controls) ; (2) NARS2 knockdown or knockout cells to confirm signal reduction; (3) Pre-absorption tests with recombinant NARS2 protein; (4) Antibody validation through immunoprecipitation coupled with mass spectrometry (IP-MS) to confirm target capture ; (5) Verification of appropriate molecular weight (50-54 kDa) in western blots . The use of recombinant antibodies with known binding epitopes can provide additional specificity advantages compared to traditional polyclonal antibodies .

What are the most common pitfalls in NARS2 antibody-based experiments?

Common pitfalls include: (1) Non-specific binding, particularly in western blot and immunofluorescence applications, which can be addressed through optimization of blocking conditions and antibody dilutions; (2) Insufficient mitochondrial permeabilization when studying this mitochondrial protein (require careful optimization of permeabilization agents); (3) Cross-reactivity with cytoplasmic asparaginyl-tRNA synthetase (NARS1), which shares sequence homology with NARS2; (4) Misinterpretation of overexpression systems, as tagged constructs may behave differently than endogenous protein; (5) Inadequate controls for validating pathogenic variants. Always include wild-type NARS2 controls when studying variant forms, and consider both positive and negative controls appropriate to the specific application .

How should researchers analyze and interpret NARS2 expression data across different tissues?

When analyzing NARS2 expression across tissues, consider: (1) Normalization strategy: Use appropriate housekeeping proteins as internal controls, preferably multiple references for robust normalization; (2) Tissue-specific variation: NARS2 expression naturally varies across tissues, with higher expression typically observed in metabolically active tissues like liver, brain, kidney, and heart ; (3) Subcellular fractionation effects: Since NARS2 is mitochondrial, expression comparisons should account for mitochondrial content differences between tissues; (4) Statistical analysis: Apply appropriate statistical tests (ANOVA with post-hoc tests for multi-tissue comparisons) and report both biological and technical replicates; (5) Quantification method: For immunohistochemistry/immunofluorescence, use standardized scoring systems (H-score or integrated optical density) for semi-quantitative analysis rather than subjective assessment.

What approaches should be used to analyze the pathogenicity of novel NARS2 variants?

For novel NARS2 variants, implement a multi-faceted approach: (1) In silico prediction tools to assess potential pathogenicity; (2) Structural modeling to predict effects on protein secondary structure ; (3) Functional studies examining expression levels, mitochondrial localization, and dimerization capacity ; (4) For potential splicing variants, perform mRNA analysis and potentially minigene experiments ; (5) Segregation analysis in family members when available ; (6) For missense variants, molecular dynamics simulations to evaluate effects on protein stability and dimer formation energy . Integration of these approaches provides stronger evidence for pathogenicity classification than any single method alone. For variants affecting conserved residues across species, evolutionary conservation analysis provides additional supporting evidence.

How can NARS2 antibodies be used to investigate mitochondrial disease mechanisms?

NARS2 antibodies can serve as powerful tools for investigating mitochondrial disease mechanisms through several approaches: (1) Tissue profiling: Characterize NARS2 expression patterns in patient samples with mitochondrial disorders versus controls; (2) Biochemical complex analysis: Use NARS2 antibodies alongside antibodies against respiratory chain complexes to assess correlation between NARS2 deficiency and specific complex deficiencies; (3) Stress response studies: Examine changes in NARS2 localization or expression under mitochondrial stress conditions; (4) Interaction studies: Identify NARS2 protein interaction partners through co-immunoprecipitation coupled with mass spectrometry; (5) Post-translational modification analysis: Investigate how modifications of NARS2 may regulate its function under physiological and pathological conditions. These approaches can reveal mechanisms connecting NARS2 dysfunction to broader mitochondrial pathology .

What methodologies are recommended for studying the impact of NARS2 mutations on mitochondrial translation?

To investigate how NARS2 mutations affect mitochondrial translation: (1) Pulse-labeling experiments using 35S-methionine in the presence of cytoplasmic translation inhibitors to specifically assess mitochondrial protein synthesis rates; (2) Northern blot analysis of mitochondrial tRNAs to assess aminoacylation status; (3) Polysome profiling to evaluate mitochondrial ribosome assembly and function; (4) Oxygen consumption rate measurements to assess functional consequences on oxidative phosphorylation; (5) Blue Native PAGE to analyze respiratory chain complex assembly. When comparing mutant and wild-type NARS2, ensure equivalent expression levels or account for expression differences in the analysis . Additionally, consider rescue experiments where wild-type NARS2 is reintroduced into cells harboring NARS2 mutations to confirm causality of observed defects.