RARS2 Antibody, HRP conjugated

What is RARS2 and why is it significant in mitochondrial research?

RARS2 encodes the mitochondrial arginyl-transfer RNA synthetase, an essential enzyme for mitochondrial protein synthesis. This protein plays a crucial role in charging tRNAs with arginine in the mitochondria, thereby facilitating translation of mitochondrially-encoded proteins. Mutations in RARS2 have been associated with pontocerebellar hypoplasia type 6 (PCH6), an early-onset encephalopathy . Research has demonstrated that RARS2 deficiency can lead to severe mitochondrial respiratory chain deficiencies involving complexes I, III, and IV, highlighting its significance in maintaining proper mitochondrial function .

Methodology for studying RARS2 typically involves:

• Protein expression analysis via western blotting

• Localization studies using immunohistochemistry

• Functional assays to assess mitochondrial respiratory chain activity

What applications are most suitable for HRP-conjugated RARS2 antibodies?

HRP-conjugated RARS2 antibodies are particularly valuable for detection methods that utilize enzymatic amplification. Based on available data, these antibodies are primarily recommended for ELISA applications . The HRP conjugation provides a direct detection system without requiring secondary antibodies, which can be especially advantageous when:

• Working with limited sample volumes

• Conducting high-throughput screening

• Performing multiplexed assays where secondary antibody cross-reactivity might be problematic

• Developing diagnostic assays requiring heightened sensitivity

The specific RARS2 antibody targeting amino acids 230-578 (such as catalog number ABIN7164328) has been validated for ELISA applications with human samples .

How should researchers validate RARS2 antibody specificity?

Proper validation of RARS2 antibodies is critical, particularly when studying disease models with RARS2 mutations. A methodological approach includes:

• Positive and negative controls:

  • Positive: Tissues/cells known to express RARS2 (e.g., mitochondria-rich tissues)

  • Negative: RARS2 knockout models or cells with RARS2 silencing

• Western blot analysis:

  • Confirm single band at expected molecular weight (~65 kDa for full-length RARS2)

  • Compare protein levels between control and RARS2-deficient samples

  • Use β-Actin (or other housekeeping proteins) for normalization

• Peptide competition assays:

  • Pre-incubate antibody with immunogenic peptide (e.g., recombinant Human Probable arginine--tRNA ligase, mitochondrial protein (230-578aa))

  • Verify signal disappearance in subsequent assays

• Cross-reactivity assessment:

  • Test against related tRNA synthetases (e.g., VARS2, TRMU)

  • Verify specificity across species if cross-species reactivity is claimed

How can RARS2 antibodies help investigate mitochondrial dysfunction in pontocerebellar hypoplasia?

RARS2 mutations have been directly linked to pontocerebellar hypoplasia type 6 (PCH6), characterized by cerebellar hypoplasia, gyral immaturity, and profound neurological deficits . HRP-conjugated RARS2 antibodies can be instrumental in:

• Characterizing mitochondrial defects:

  • Quantifying RARS2 protein levels in patient-derived cells

  • Correlating RARS2 expression with respiratory chain complex abundances

  • Assessing mitochondrial morphology in conjunction with mitochondrial markers

• Analyzing tissue-specific pathology:

  • Conducting immunohistochemistry on brain tissues to map RARS2 expression patterns

  • Correlating RARS2 deficiency with neuronal loss in specific brain regions, particularly pontine nuclei

  • Examining peripheral nerve pathology in relation to RARS2 expression

• Evaluating mutation consequences:

  • Comparing RARS2 protein levels between different RARS2 mutation types

  • Assessing effects of Kozak sequence variants on protein translation

  • Determining cell-specific vulnerability to RARS2 deficiency

Recent studies have demonstrated that RARS2 mutations can result in near-global cytochrome c oxidase deficiency and severe impairment of respiratory chain complexes I, III, and IV , providing important markers for antibody-based investigations.

What methodological considerations are important when using HRP-conjugated RARS2 antibodies in immunoassays?

When employing HRP-conjugated RARS2 antibodies in research, several technical considerations can optimize results:

• Signal development parameters:

  • Substrate selection (TMB, DAB, or luminol-based systems)

  • Incubation time optimization for maximum signal-to-noise ratio

  • Signal quenching protocols to prevent oversaturation

• Blocking optimization:

  • Use of specialized blocking buffers (e.g., Odyssey™ Blocking Buffer)

  • Optimization of antibody dilution (starting recommendation: 1:16000 for western blotting)

  • Minimization of background through proper washing conditions (0.2% PBST)

• Sample preparation considerations:

  • Mitochondrial enrichment protocols for enhanced sensitivity

  • Preservation of protein integrity during extraction

  • Denaturation conditions that maintain epitope accessibility

• Detection system compatibility:

  • Imaging systems calibration (e.g., Odyssey CLx imaging system)

  • Quantification software parameters (e.g., Image Studio Lite)

  • Dynamic range assessment for accurate quantification

How do transcription and translation defects in RARS2 impact antibody-based detection methods?

Recent research has revealed complex regulatory mechanisms affecting RARS2 expression, including both transcriptional and translational control. These findings have important implications for antibody-based detection:

• Kozak sequence variants affect:

  • Researchers should be aware that variants in the Kozak sequence (e.g., c.-2A>G) dramatically reduce RARS2 protein levels despite only modestly affecting mRNA levels

  • This creates a potential disconnect between transcript quantification and protein detection

• Methodological approaches:

  • Parallel analysis of mRNA (via RT-PCR) and protein (via antibody-based methods)

  • Normalization strategies accounting for potential post-transcriptional regulation

  • Use of multiple antibodies targeting different RARS2 epitopes

• Detection sensitivity considerations:

  • Lower detection limits may be required for severely reduced RARS2 protein levels

  • Signal amplification strategies (extended substrate incubation, tyramide signal amplification)

  • Correlation with functional assays of mitochondrial activity

What are common sources of background when using HRP-conjugated RARS2 antibodies?

Optimizing signal-to-noise ratio is critical when working with HRP-conjugated antibodies. Common background sources include:

• Endogenous peroxidase activity:

  • Solution: Quench endogenous peroxidases with H₂O₂ treatment before antibody application

  • Verification: Include no-primary antibody controls

• Non-specific binding:

  • Solution: Optimize blocking conditions (protein concentration, detergent levels)

  • Verification: Include isotype controls (e.g., rabbit IgG for rabbit-host RARS2 antibodies)

• Cross-reactivity with related proteins:

  • Solution: Increase antibody dilution and washing stringency

  • Verification: Test antibody in RARS2-knockout or knockdown models

• Substrate precipitation:

  • Solution: Optimize development time and substrate concentration

  • Verification: Monitor development visually to prevent overdevelopment

How should researchers interpret RARS2 antibody signals in cells with RARS2 mutations?

Interpreting RARS2 antibody signals in mutant contexts requires careful consideration:

• Mutation-specific effects:

  • Truncation mutations may eliminate epitopes, resulting in false negatives

  • Missense mutations might affect epitope structure without eliminating signal

  • Kozak sequence variants dramatically reduce protein levels despite modest mRNA changes

• Subcellular localization assessment:

  • Some mutations may alter RARS2 localization rather than abundance

  • Co-staining with mitochondrial markers recommended

  • Differential extraction protocols to distinguish mislocalized protein

• Quantification approaches:

  • Normalization to housekeeping proteins (e.g., β-Actin)

  • Comparison to wild-type controls

  • Calibration curves with recombinant protein standards

How can RARS2 antibodies contribute to understanding respiratory chain deficiencies?

RARS2 mutations have been linked to severe mitochondrial respiratory chain deficiencies. HRP-conjugated RARS2 antibodies can help elucidate these mechanisms:

• Correlation analysis:

  • Compare RARS2 levels with respiratory chain complex abundances

  • Assess relationship between RARS2 deficiency and COX (cytochrome c oxidase) activity

  • Evaluate mitochondrial translation products in relation to RARS2 expression

• Tissue-specific investigations:

  • Map respiratory chain deficiencies across tissues with varying RARS2 expression

  • Focus on particularly affected tissues (brain, heart, muscle)

  • Compare findings with other mitochondrial translation defects

• Therapeutic intervention assessment:

  • Monitor RARS2 levels during potential therapies

  • Correlate RARS2 restoration with respiratory chain function

  • Identify compensation mechanisms in partial RARS2 deficiency

Studies have revealed that RARS2 mutations can cause "near-global cytochrome c oxidase-deficiency" with "severe deficiencies involving complexes I, III, and IV" , providing important targets for antibody-based quantification.

What is the relationship between RARS2 and other mitochondrial RNA processing factors?

Interestingly, RARS2 shows expression patterns similar to other mitochondrial RNA processing factors, suggesting coordinated regulation:

• Co-expression relationships:

  • RARS2 expression correlates with other mitochondrial tRNA synthetases (VARS2, TRMU)

  • Expression patterns parallel mitochondrial translation factors (MTG1, MTIF3)

  • Similar regulation as mitoribosomal proteins

• Investigation approaches:

  • Multiplex staining with antibodies against related factors

  • Co-immunoprecipitation to identify protein interactions

  • Parallel knockdown studies to assess functional relationships

• Mitochondrial stress responses:

  • Monitor RARS2 in relation to other factors during mitochondrial stress

  • Assess expression changes in response to mtDNA depletion

  • Evaluate compensatory mechanisms