HARS2 Antibody

What is the function of HARS2 and why is it significant in mitochondrial research?

HARS2 encodes mitochondrial histidyl-tRNA synthetase, which is crucial for mitochondrial protein synthesis by catalyzing the attachment of histidine to its cognate tRNA in the mitochondrial matrix. This process is an essential component of mitochondrial translation machinery. The significance of HARS2 extends beyond normal cellular function to disease states, as biallelic variants in HARS2 are associated with Perrault syndrome, a rare autosomal recessive disorder characterized by sensorineural hearing loss in both sexes and primary ovarian insufficiency in females with 46,XX karyotype. Some patients also develop neurological manifestations including peripheral neuropathy, cerebellar ataxia, and intellectual disability .

Research into HARS2 provides valuable insights into mitochondrial function, aminoacyl-tRNA synthetase biology, and the pathophysiological mechanisms underlying Perrault syndrome and other related disorders.

Which methods are most effective for HARS2 protein detection?

Several methodologies can be employed for detecting HARS2 protein in experimental settings:

• Western Blotting (WB): This is the most commonly used technique for HARS2 detection. Multiple validated antibodies have demonstrated successful detection in various human samples (MCF7 cells, HEK-293 cells, heart tissue) and mouse kidney tissue .

• Immunohistochemistry (IHC): HARS2 can be visualized in tissue sections using specific antibodies. Successful detection has been reported in human breast cancer tissue and kidney tissue, with optimal results achieved using TE buffer pH 9 for antigen retrieval .

• Immunoprecipitation (IP): Effective for isolating HARS2 protein complexes, with positive results demonstrated in HEK-293 cells .

• ELISA: Useful for quantitative analysis of HARS2 protein levels in various sample types .

• Flow Cytometry (FACS): Some antibodies have been validated for flow cytometry applications, allowing analysis of HARS2 in individual cells .

The selection of an appropriate detection method should be guided by your specific research question, sample type, and required sensitivity.

What criteria should guide the selection of a HARS2 antibody?

When selecting a HARS2 antibody, consider these critical factors:

• Target epitope: HARS2 antibodies target different amino acid regions (e.g., AA 34-506, AA 2-110, AA 383-432, N-terminus, or AA 217-506). Choose an epitope region based on your research focus, considering whether you need to detect specific domains or variants .

• Host species: Available options include rabbit (polyclonal) and mouse (monoclonal and polyclonal). Select based on compatibility with other antibodies in multiplex experiments and secondary detection systems .

• Validated applications: Ensure the antibody is validated for your intended application (WB, IHC, IP, ELISA, or FACS). Review published validation data for the specific application .

• Cross-reactivity: Consider whether cross-reactivity with species of interest (human, mouse, rat, dog, monkey) is needed or should be avoided .

• Clonality: Monoclonal antibodies offer higher specificity but may miss isoforms or variants. Polyclonal antibodies provide broader detection but potentially higher background .

• Conjugation: Determine if you need unconjugated antibody or one conjugated to enzymes (HRP), fluorophores (FITC), or affinity tags (biotin) based on your detection system .

What are the optimal sample preparation methods for HARS2 detection?

Effective sample preparation is crucial for reliable HARS2 detection:

• Cell/Tissue Lysis: For mitochondrial proteins like HARS2, use lysis buffers containing 1-2% non-ionic detergents (Triton X-100 or NP-40) supplemented with protease inhibitors. Mitochondrial isolation protocols may improve signal-to-noise ratio.

• Protein Quantification: Bradford or BCA assays are recommended to ensure equal loading of samples.

• Denaturation: Heat samples at 95°C for 5 minutes in Laemmli buffer containing 5% β-mercaptoethanol for optimal denaturation.

• Tissue Preparation for IHC: Formalin-fixed paraffin-embedded sections typically require antigen retrieval with TE buffer at pH 9 for optimal HARS2 detection .

• Cell Preparation for Flow Cytometry: Permeabilization with 0.1% saponin or 0.1% Triton X-100 is essential for accessing this intracellular mitochondrial protein.

Validated positive controls include MCF7 cells, HEK-293 cells, human heart tissue, and mouse kidney tissue, which have demonstrated reliable HARS2 expression .

How can HARS2 antibodies be utilized to investigate protein-protein interactions?

Investigating HARS2 protein interactions requires sophisticated methodological approaches:

• Co-immunoprecipitation (Co-IP): Use HARS2 antibodies to pull down protein complexes from cellular lysates followed by Western blotting for potential interacting partners. HEK-293 cells have been successfully used for HARS2 immunoprecipitation .

• Proximity Ligation Assay (PLA): This technique allows visualization of protein interactions in situ with subcellular resolution, crucial for determining if interactions occur within mitochondria.

• Förster Resonance Energy Transfer (FRET): Combine HARS2 antibodies labeled with donor fluorophores with suspected interaction partners labeled with acceptor fluorophores to measure energy transfer indicative of physical proximity.

• Cross-linking Mass Spectrometry (XL-MS): Cross-link protein complexes in intact mitochondria before immunoprecipitation with HARS2 antibodies and analysis by mass spectrometry.

HARS2 likely functions as a homodimer and contains domains for histidine binding, dimer interaction, and tRNA binding . The protein's structural features, particularly the predicted dimer interface region containing conserved residues like Arg138, are important considerations when designing interaction experiments.

What approaches can resolve contradictory findings in HARS2 expression studies?

Resolving contradictory results in HARS2 expression studies requires systematic troubleshooting:

• Antibody Validation Panel: Test multiple antibodies targeting different epitopes of HARS2. Compare antibodies recognizing different regions (N-terminal vs. C-terminal) as post-translational modifications or mutations might affect epitope accessibility .

• Knockout Controls: Generate CRISPR/Cas9 HARS2 knockout cell lines as negative controls to confirm antibody specificity.

• Transcript-Protein Correlation: Compare protein detection (Western blot) with transcript levels (RT-qPCR) to identify discrepancies between transcription and translation.

• Subcellular Fractionation: Isolate mitochondrial fractions to enrich for HARS2 and reduce cytoplasmic contamination that may lead to inconsistent results.

• Multiple Detection Methods: Cross-validate findings using different techniques (WB, IHC, IP) as each has different sensitivity and specificity profiles .

When published results conflict, consider that expression may vary by cell type, developmental stage, or in response to specific stressors affecting mitochondrial function.

How can HARS2 antibodies be employed to study Perrault syndrome pathophysiology?

Investigating Perrault syndrome using HARS2 antibodies requires specialized experimental approaches:

• Patient-Derived Cell Models: Compare HARS2 protein levels, subcellular localization, and stability in fibroblasts or induced pluripotent stem cells (iPSCs) from patients with different HARS2 variants versus controls.

• Variant-Specific Analysis: Use antibodies targeting regions containing known pathogenic variants (such as p.Arg480His) to assess potential differences in detection efficiency that might reflect structural changes .

• Functional Readouts: Combine HARS2 immunodetection with assays measuring mitochondrial translation efficiency, ATP production, or respiratory chain complex assembly to correlate protein expression with functional outcomes.

• Tissue-Specific Studies: As Perrault syndrome affects specific tissues (inner ear, ovaries), use immunohistochemistry to examine HARS2 expression patterns in relevant tissues from appropriate model systems .

• Interactome Analysis: Compare HARS2 protein interactions in normal versus disease models to identify pathways disrupted by pathogenic variants.

Research has identified several pathogenic variants in HARS2, including the recurrent variant c.1439G>A (p.Arg480His), which appears in compound heterozygosity with other variants in affected individuals from three unrelated families with sensorineural hearing loss .

What methodological approaches can detect conformational changes in HARS2 protein?

Detecting conformational changes in HARS2 requires specialized techniques:

• Conformation-Specific Antibodies: Develop or identify antibodies that specifically recognize different conformational states of HARS2, particularly around the histidine binding pocket or dimer interface.

• Limited Proteolysis Combined with Western Blotting: Different conformations exhibit different protease accessibility patterns; digest HARS2 under various conditions and detect fragments with domain-specific antibodies .

• Hydrogen-Deuterium Exchange Mass Spectrometry (HDX-MS): This technique can reveal regions of HARS2 with altered solvent accessibility when combined with immunoprecipitation using HARS2 antibodies.

• Crosslinking Approaches: Chemical crosslinking can "freeze" protein conformations, which can then be analyzed by Western blotting to detect altered migration patterns indicative of conformational changes.

• Fluorescence-Based Thermal Shift Assays: When combined with immunoprecipitated HARS2, these assays can detect ligand-induced or mutation-induced stability changes.

HARS2 contains distinct domains for histidine binding, dimer interaction, and tRNA binding . Pathogenic variants like p.Arg138His may interfere with salt bridge formation and subsequently affect dimer interaction and active site conformation, while variants like p.Arg480His in the C-terminal domain may disrupt tRNA recognition and binding .

How can researchers optimize HARS2 detection in challenging tissue samples?

Optimizing HARS2 detection in challenging samples requires advanced troubleshooting:

• Antigen Retrieval Optimization: For formalin-fixed tissues, systematic testing of retrieval conditions is essential. TE buffer at pH 9 has been specifically recommended for HARS2 IHC .

• Signal Amplification Systems: Consider tyramide signal amplification (TSA) or polymer-based detection systems to enhance sensitivity while maintaining specificity.

• Dual Antibody Approach: Simultaneously use two primary antibodies targeting different HARS2 epitopes (e.g., N-terminal and C-terminal) with differently labeled secondary antibodies to confirm specificity.

• Background Reduction Strategies: For tissues with high mitochondrial content (heart, kidney), use specialized blocking buffers containing both protein blockers and non-immune serum from the secondary antibody host species.

• Tissue-Specific Protocol Modifications:

  • Brain tissue: Extended fixation periods may require longer antigen retrieval

  • Adipose tissue: Additional delipidation steps

  • Highly vascularized tissues: Quenching of endogenous peroxidase activity

  • Fibrotic tissues: Addition of protein denaturants to improve epitope accessibility

• Multiplexing with Mitochondrial Markers: Co-staining with established mitochondrial markers (e.g., TOMM20) can help distinguish true HARS2 signal from background.

What strategies enable quantitative analysis of HARS2 expression across different experimental conditions?

For rigorous quantitative analysis of HARS2 expression:

How can researchers distinguish between HARS2 variants using antibody-based methods?

Distinguishing HARS2 variants requires sophisticated approaches:

• Variant-Specific Antibodies: Develop antibodies specifically raised against peptides containing variant residues. For example, antibodies that can distinguish between wild-type Arg480 and the pathogenic His480 variant .

• Post-IP Analysis: Immunoprecipitate HARS2 using a general antibody, followed by mass spectrometry to identify specific variants based on peptide mass differences.

• Mobility Shift Assays: Some variants may cause subtle changes in migration patterns on SDS-PAGE that can be detected using high-percentage gels or Phos-tag gels (if phosphorylation is affected).

• Combination with Genetic Analysis: Couple antibody detection with techniques like allele-specific PCR to correlate protein detection with specific genetic variants.

• Structural Epitope Mapping: Use a panel of antibodies targeting different regions to identify structural changes in variant proteins that may alter epitope accessibility.

Known pathogenic variants in HARS2 include p.Leu200Val, p.Val368Leu, p.Tyr337Cys, p.Lys58Glu, p.Arg150Cys, p.Arg327Gln, p.Leu46Gln, p.Arg87Cys, p.Arg138His, and the recurrent p.Arg480His variant . Each may have distinct effects on protein structure and function that could potentially be distinguished using appropriate antibody-based approaches.

What are the most common technical challenges when working with HARS2 antibodies?

Researchers commonly encounter these challenges when working with HARS2 antibodies:

• Mitochondrial Localization Issues: The mitochondrial localization of HARS2 can present challenges for antibody accessibility, particularly in fixed tissues.

• Cross-Reactivity with Cytosolic HARS: Due to ~73% sequence homology between mitochondrial HARS2 and nuclear HARS, antibody cross-reactivity must be carefully evaluated .

• Isoform Detection: Ensure the selected antibody can detect all relevant HARS2 isoforms or specifically distinguish between them if needed.

• Fixation Sensitivity: Overfixation can mask epitopes, particularly in mitochondrial proteins. Optimize fixation protocols for each application.

• Low Abundance in Some Tissues: HARS2 may be expressed at low levels in certain tissues, requiring signal amplification strategies.

Troubleshooting approaches include:

• Testing multiple antibodies targeting different epitopes

• Including appropriate positive controls (MCF7 cells, HEK-293 cells, human heart tissue)

• Optimizing protein extraction protocols specifically for mitochondrial proteins

• Validating specificity using genetic knockdown approaches

How can researchers validate antibody specificity for HARS2 in different experimental contexts?

Comprehensive validation of HARS2 antibody specificity should include:

• Genetic Controls:

  • CRISPR/Cas9 knockout cell lines

  • siRNA or shRNA knockdown

  • Overexpression systems using tagged HARS2 constructs

• Peptide Competition Assays: Pre-incubation of the antibody with the immunizing peptide should abolish specific signal.

• Multi-antibody Approach: Compare staining patterns using antibodies targeting different regions of HARS2 (N-terminal, middle region, C-terminal) .

• Mass Spectrometry Validation: Immunoprecipitate with the HARS2 antibody and confirm protein identity by mass spectrometry.

• Tissue/Cell Type Controls:

  • Positive controls: MCF7 cells, HEK-293 cells, human heart tissue, mouse kidney tissue

  • Negative controls: Tissues known to express minimal HARS2

  • Species cross-reactivity testing if working with non-human models

• Application-Specific Validation:

  • For WB: Test multiple negative controls and analyze at the expected molecular weight

  • For IHC: Include isotype controls and test multiple fixation and antigen retrieval conditions

  • For IP: Confirm enrichment of HARS2 in the immunoprecipitated fraction versus input

How can HARS2 antibodies contribute to understanding mitochondrial stress responses?

HARS2 antibodies can provide valuable insights into mitochondrial stress responses through:

• Stress-Induced Localization Changes: Track HARS2 subcellular redistribution during mitochondrial stress using immunofluorescence microscopy with co-localization analysis.

• Post-Translational Modification Analysis: Combine HARS2 immunoprecipitation with mass spectrometry or modification-specific antibodies to identify stress-induced modifications.

• Protein Stability Assessment: Monitor HARS2 protein levels during mitochondrial stress conditions using quantitative Western blotting to determine if degradation pathways are activated.

• Mitochondrial Translation Efficiency: Correlate HARS2 levels with mitochondrial protein synthesis rates during stress by combining immunoblotting with metabolic labeling of mitochondrial translation products.

• Tissue-Specific Responses: Compare HARS2 expression patterns across different tissues following systemic stressors using immunohistochemistry to identify differential vulnerability.

• Interaction Dynamics: Examine how HARS2 protein-protein interactions change during mitochondrial stress using co-immunoprecipitation followed by Western blotting or mass spectrometry.

This approach is particularly relevant given HARS2's essential role in mitochondrial translation and the association of HARS2 mutations with tissue-specific pathologies in Perrault syndrome .

What methodological considerations apply when studying HARS2 in model organisms?

When investigating HARS2 in model organisms, consider these methodological aspects:

• Antibody Cross-Reactivity: Verify cross-reactivity of human HARS2 antibodies with the species of interest. Some antibodies have confirmed reactivity with mouse and rat HARS2 .

• Evolutionary Conservation Analysis: HARS2 shares ~23% sequence homology with the E. coli ortholog HisRS . Consider epitope conservation when selecting antibodies for use in evolutionary studies.

• Tissue-Specific Expression Patterns:

  • Develop standardized IHC protocols for each model organism and tissue type

  • Create expression atlases across different developmental stages and tissues

• Genetic Model Validation:

  • For knockout models: Confirm absence of protein with antibodies

  • For knockin models of human variants: Verify protein expression and localization

• Species-Specific Controls:

  • Include wild-type and knockout tissues from the same species

  • Consider fixation differences between species when optimizing IHC protocols

• Functional Readouts:

  • Develop species-appropriate assays for mitochondrial translation efficiency

  • Adapt hearing and ovarian function assessments for models of Perrault syndrome

The use of model organisms is particularly valuable for studying Perrault syndrome pathophysiology, as it allows investigation of tissue-specific effects of HARS2 variants on both hearing and ovarian function .