RARS2 Antibody

What is RARS2 and what is its primary function in cellular metabolism?

RARS2 is a mitochondrial arginyl-transfer RNA synthetase that plays a crucial role in mitochondrial protein translation. It catalyzes a two-step reaction: first, arginine is activated by ATP to form Arg-AMP, and then it's transferred to the acceptor end of tRNA(Arg) . This process is essential for proper mitochondrial protein synthesis and energy production. RARS2 is encoded by the nuclear genome but functions within mitochondria, making it crucial for mitochondrial function. Biallelic pathogenic variants in the RARS2 gene lead to mitochondrial encephalopathy, characterized by severe neurological deficits .

What experimental applications are RARS2 antibodies validated for?

Based on available data, RARS2 antibodies have been validated for several experimental applications:

Researchers should note that these applications have been specifically tested with antibodies like ab230274, and validation for untested applications should be performed before proceeding with extensive experiments .

What species reactivity has been confirmed for RARS2 antibodies?

RARS2 antibodies have been confirmed to react with human and mouse samples . This cross-reactivity is based on sequence homology and experimental validation. When working with other species, preliminary validation experiments are strongly recommended, as cross-reactivity cannot be guaranteed based solely on sequence similarity. For species with high homology to human RARS2, such as non-human primates, the antibodies may work but require experimental confirmation.

How can researchers optimize RARS2 antibody protocols for studying mitochondrial dysfunction in disease models?

When studying mitochondrial dysfunction using RARS2 antibodies, consider these optimization strategies:

• Subcellular fractionation: Isolate mitochondrial fractions before Western blotting to enrich for RARS2 protein and reduce background signal.

• Co-localization studies: Combine RARS2 antibodies with established mitochondrial markers (e.g., TOMM20, COX4) for immunofluorescence microscopy to confirm mitochondrial localization.

• Patient-derived models: When studying RARS2-related disorders, patient fibroblasts or iPSC-derived neurons can be valuable models, as demonstrated in studies of mitochondrial encephalopathies .

• Metabolomic correlation: Correlate RARS2 expression patterns with metabolomic changes, particularly focusing on lysophospholipid and sphingomyelin-related metabolites that have been associated with RARS2 dysfunction .

• Oxidative phosphorylation assessment: Combine RARS2 antibody studies with functional assessments of mitochondrial respiration to establish phenotype-genotype correlations.

What are the methodological considerations when using RARS2 antibodies to study pontocerebellar hypoplasia type 6?

When studying PCH6 using RARS2 antibodies, researchers should consider:

How can researchers differentiate between wild-type and mutant RARS2 proteins using antibody-based techniques?

Differentiating wild-type from mutant RARS2 can be challenging but is possible with these approaches:

• Variant-specific antibodies: For common mutations that result in amino acid changes, custom antibodies can be developed that specifically recognize the mutant epitope.

• Molecular weight shifts: Some mutations may cause detectable size differences that can be resolved on high-percentage or gradient SDS-PAGE gels.

• Functional assays: Combine antibody detection with aminoacylation activity assays to assess functional differences between wild-type and mutant proteins.

• Subcellular localization: Some mutations may disrupt mitochondrial targeting signals, resulting in altered localization that can be detected by immunofluorescence or subcellular fractionation.

• Protein stability studies: Pulse-chase experiments using cycloheximide can reveal differences in protein stability between wild-type and mutant RARS2.

What approaches can be used to study RARS2 expression in patient-derived induced pluripotent stem cells (iPSCs)?

For RARS2 studies in iPSCs, consider these methodological approaches:

• Neural differentiation protocols: Differentiate iPSCs into neurons or astrocytes to study cell-type-specific effects of RARS2 mutations, as has been done for other mitochondrial disorders .

• Temporal expression analysis: Monitor RARS2 expression during different stages of neural differentiation using Western blotting and immunofluorescence.

• Drug screening platforms: Use patient-derived iPSCs for high-throughput screening of drug libraries, such as the Prestwick Chemical Library, to identify compounds that may rescue RARS2-related phenotypes .

• CRISPR-Cas9 correction: Generate isogenic control lines by correcting RARS2 mutations to establish causality and study rescue effects.

• Co-culture systems: Develop co-cultures of neurons and astrocytes to study non-cell-autonomous effects of RARS2 dysfunction.

What is the optimal protocol for extracting and preserving RARS2 protein for Western blot analysis?

For optimal RARS2 protein extraction and preservation:

• Lysis buffer composition: Use a mitochondria-friendly lysis buffer containing 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1% Triton X-100, 1 mM EDTA, with protease inhibitor cocktail.

• Homogenization technique: For tissue samples, use a Dounce homogenizer with 10-15 strokes to ensure complete lysis without protein degradation.

• Temperature considerations: Maintain samples at 4°C throughout extraction to prevent degradation.

• Reducing agents: Include 1-5 mM DTT or β-mercaptoethanol in sample buffer to maintain reducing conditions.

• Storage conditions: Aliquot lysates and store at -80°C to avoid freeze-thaw cycles that can degrade RARS2.

• Loading controls: Include mitochondrial markers (e.g., VDAC1) alongside standard loading controls (e.g., ACTB) when analyzing RARS2 by Western blot .

What controls are essential when validating RARS2 antibody specificity in experimental systems?

Essential controls for RARS2 antibody validation include:

• Positive controls: Use samples known to express RARS2, such as HL-60 cell lysates or mouse kidney tissue lysates .

• Negative controls: Include samples where RARS2 is knocked down (siRNA) or knocked out (CRISPR) to confirm specificity.

• Peptide competition: Pre-incubate the antibody with the immunizing peptide to demonstrate specific binding.

• Secondary antibody-only controls: Omit primary antibody to assess background from secondary antibody.

• Cross-reactivity assessment: Test the antibody against recombinant RARS1 (cytosolic homolog) to ensure specificity for the mitochondrial isoform.

• Multiple antibody validation: When possible, confirm results using antibodies targeting different epitopes of RARS2.

How should researchers optimize immunohistochemistry protocols for RARS2 detection in tissue sections?

For optimal RARS2 immunohistochemistry:

• Fixation method: Use 10% neutral-buffered formalin for 24-48 hours, as overfixation may mask epitopes.

• Antigen retrieval: Heat-mediated antigen retrieval in citrate buffer (pH 6.0) is typically effective for RARS2 epitope exposure.

• Blocking conditions: Block with 5-10% normal serum from the species of the secondary antibody for 1 hour at room temperature.

• Antibody dilution: Start with 1:100 dilution for RARS2 antibody, as used successfully for human lung and gastric cancer tissues .

• Incubation conditions: Incubate primary antibody overnight at 4°C to maximize specific binding while minimizing background.

• Detection system: DAB (3,3'-diaminobenzidine) or AEC (3-amino-9-ethylcarbazole) systems are both compatible with RARS2 detection.

• Counterstaining: Light hematoxylin counterstaining provides good nuclear contrast without obscuring RARS2 signal.

What are the best approaches for quantifying RARS2 mRNA expression levels in patient samples?

For quantifying RARS2 mRNA expression:

• Sample collection: For blood samples, use RNA preservation tubes to maintain RNA integrity.

• RNA extraction: Use specialized kits like miRNeasy for high-quality RNA extraction from peripheral blood or tissues .

• Reverse transcription: Convert 150-200 ng of total RNA to cDNA using reliable reverse transcription kits.

• Reference gene selection: Use stable reference genes such as ACTB for normalization, but validate stability across your experimental conditions .

• Primer design: Design primers spanning exon-exon junctions to avoid genomic DNA amplification.

• Quantification method: Use SYBR green or probe-based qPCR with technical triplicates for each sample .

• Data analysis: Apply the ΔΔCt method for relative quantification, or use standard curves for absolute quantification.

How can researchers apply RARS2 antibodies in genome-wide functional studies?

RARS2 antibodies can be integrated into genome-wide functional studies through:

• ChIP-seq applications: Although not a transcription factor, RARS2 may interact with DNA-binding proteins, making ChIP-seq a potential application for studying its genomic interactions.

• Protein interactome analysis: Use RARS2 antibodies for immunoprecipitation followed by mass spectrometry to identify protein interaction networks.

• CRISPR screen validations: After genome-wide CRISPR screens like those described for host dependency factors , RARS2 antibodies can validate the effects of genetic perturbations on protein expression.

• Proteomics integration: Combine RARS2 antibody-based assays with proteomic approaches to create comprehensive datasets for meta-analysis similar to the MAIC approach described for influenza studies .

• High-content imaging: Use RARS2 antibodies in high-content screening assays to assess mitochondrial phenotypes across genetic or chemical perturbations.

How can RARS2 antibodies be used to study novel clinical phenotypes beyond the classical presentation?

Recent research has expanded the clinical spectrum of RARS2-related disorders beyond the classical presentation. Researchers can use RARS2 antibodies to:

• Phenotype correlation studies: Compare RARS2 expression and localization in tissues from patients with classical versus novel phenotypes, such as the recently described Lennox-Gastaut Syndrome without pontocerebellar hypoplasia .

• Brain region specificity: Analyze RARS2 expression across different brain regions to understand the regional vulnerability patterns that may explain diverse neurological presentations.

• Developmental expression: Track RARS2 expression during neurodevelopment to identify critical windows that may explain phenotypic variability.

• Genotype-phenotype correlation: Compare RARS2 expression patterns between patients with different mutations, including novel promoter variants that affect expression rather than protein structure .

• Tissue-specific effects: Investigate why certain RARS2 mutations affect specific tissues despite ubiquitous expression, using tissue microarrays and RARS2 antibodies.

What are the methodological considerations for studying RARS2 in the context of metabolomic alterations?

When integrating RARS2 antibody studies with metabolomics:

• Sample preparation compatibility: Design protocols that allow parallel processing of samples for both protein analysis and metabolite extraction.

• Correlation analyses: Establish statistical frameworks to correlate RARS2 protein levels with specific metabolite changes, particularly focusing on lysophospholipid and sphingomyelin-related metabolites .

• Time-course studies: Design experiments that capture dynamic changes in both RARS2 expression and metabolite profiles.

• Subcellular metabolomics: Combine organelle isolation techniques with metabolomics to specifically analyze mitochondrial metabolites in relation to RARS2 expression.

• Stable isotope tracing: Use stable isotope-labeled amino acids to track arginine metabolism and tRNA charging in relation to RARS2 function and expression.

How can researchers utilize RARS2 antibodies in therapeutic development for mitochondrial disorders?

For therapeutic development applications:

• High-throughput screening: Develop cell-based assays using RARS2 antibodies to screen drug libraries, such as the Prestwick Chemical Library containing over 1200 FDA-approved compounds .

• Therapeutic monitoring: Use RARS2 antibodies to assess whether therapeutic interventions restore normal RARS2 expression, localization, or function.

• Patient stratification: Develop standardized RARS2 antibody-based assays to categorize patients based on protein expression patterns for clinical trials.

• Gene therapy validation: Use RARS2 antibodies to confirm successful gene replacement or correction strategies.

• Biomarker development: Investigate whether RARS2 protein levels in accessible tissues correlate with disease severity or progression, potentially serving as biomarkers.