TARS2 Antibody

What is TARS2 and why is it important in research?

TARS2 encodes the mitochondrial threonyl-tRNA synthetase, an essential enzyme responsible for aminoacylation of threonyl-tRNA in mitochondria. This protein plays a critical role in mitochondrial protein synthesis by ensuring proper charging of tRNA with threonine. TARS2 has gained research importance due to its association with mitochondrial disorders, neurodevelopmental phenotypes, and potential involvement in various cancers . Mutations in TARS2 have been linked to multiple mitochondrial respiratory chain (MRC) defects, axial hypotonia, and severe psychomotor delay, making it an important target for researchers investigating mitochondrial diseases .

What are the best methods for validating TARS2 antibody specificity?

Validating TARS2 antibody specificity requires multiple complementary approaches. First, perform Western blotting using both wild-type samples and those with known TARS2 deficiency to confirm the presence of bands at the expected molecular weight (~80 kDa). Second, implement a genetic knockout/knockdown system to demonstrate absence or reduction of signal. Third, conduct immunoprecipitation followed by mass spectrometry to confirm that the antibody is capturing TARS2 protein. Finally, employ immunofluorescence to verify the expected mitochondrial localization pattern, which should co-localize with established mitochondrial markers .

How should TARS2 antibodies be used in Western blot applications?

For optimal TARS2 detection in Western blot applications, extract proteins from approximately 10^6 cells using sonication and solubilization techniques as described in mitochondrial protein protocols. Load 50 μg protein per lane on SDS-polyacrylamide gels. Use commercially available antibodies against TARS2 (such as those from GeneTex) at the manufacturer's recommended dilution, with HSP60 (Abcam) as a mitochondrial loading control . For visualization, employ either chemiluminescence or fluorescence-based secondary antibodies. Expect to observe varying TARS2 protein levels depending on the tissue type, with particular attention to potential decreases in cells harboring TARS2 mutations.

What are the key considerations when selecting control samples for TARS2 antibody experiments?

Selection of appropriate controls is critical for TARS2 antibody experiments. Include positive controls from tissues known to express TARS2 (fibroblasts, neuronal tissues, or liver samples work well). For negative controls, use samples with confirmed TARS2 deficiency or knockdown. When studying patient samples with suspected TARS2 mutations, age-matched and tissue-matched controls are essential, as TARS2 expression patterns may vary developmentally and across tissue types. Additionally, include technical controls such as secondary-antibody-only samples to detect non-specific binding. For comparative studies, HSP60 or other mitochondrial proteins serve as reliable loading controls to normalize TARS2 expression levels .

How can TARS2 antibodies be utilized to investigate its potential role in the mTORC1 signaling pathway?

Recent research suggests TARS2 may interact with the mTORC1 signaling pathway, particularly through binding to Rag GTPases . To investigate this relationship, researchers should employ co-immunoprecipitation assays using TARS2 antibodies to pull down protein complexes, followed by Western blotting for mTORC1 components (mTOR, Raptor) and Rag GTPases. Proximity ligation assays can visualize TARS2-mTORC1 interactions in situ. Immunofluorescence co-localization studies should examine TARS2, mTORC1 components, and lysosomes (where mTORC1 is activated). For functional analysis, combine TARS2 antibody-based detection with phospho-specific antibodies against downstream mTORC1 targets (p70S6K, 4E-BP1) in cells with wild-type versus mutant TARS2 (particularly variants within the 301-381 region) to assess pathway activation .

What methodological approaches can differentiate between canonical aminoacylation functions and non-canonical roles of TARS2?

Differentiating between TARS2's canonical aminoacylation function and potential non-canonical roles requires sophisticated experimental design. First, employ tRNA aminoacylation assays using acid-urea PAGE followed by Northern blotting with radiolabeled probes to quantify charged versus uncharged tRNA^Thr levels . This establishes baseline aminoacylation activity. To identify non-canonical functions, perform immunoprecipitation with TARS2 antibodies followed by mass spectrometry to identify interaction partners beyond the translation machinery. Conduct subcellular fractionation followed by Western blotting to determine if TARS2 localizes to unexpected compartments. Use domain-specific TARS2 antibodies to map which regions participate in novel interactions. Finally, employ CRISPR-Cas9 to create separation-of-function mutants that maintain aminoacylation but disrupt other activities, then use TARS2 antibodies to track protein localization and interaction changes.

How can TARS2 antibodies be used to investigate the differential expression and amplification of TARS2 in cancer tissues?

Investigating TARS2 amplification in cancer requires a multi-pronged approach using TARS2 antibodies. First, perform immunohistochemistry on tissue microarrays containing multiple cancer types alongside matched normal tissues to quantify expression patterns. Use TARS2 antibodies in conjunction with digital pathology analysis to establish H-scores or other quantitative metrics. For mechanistic studies, combine fluorescence in situ hybridization (FISH) to detect gene amplification with immunofluorescence using TARS2 antibodies to correlate copy number with protein expression at the single-cell level. Western blot analysis of cancer cell lines with known TARS2 amplification status can establish whether copy number gains translate to increased protein levels . Finally, chromatin immunoprecipitation followed by sequencing (ChIP-seq) using antibodies against transcription factors predicted to regulate TARS2 can identify upstream mechanisms driving overexpression.

What are the technical challenges in detecting TARS2 protein in patient-derived samples with intronic mutations?

Detecting TARS2 protein in patients with intronic mutations presents significant technical challenges. Intronic mutations, particularly those affecting splicing (such as the c.695+3A>G mutation), often result in aberrant and highly unstable transcripts . When analyzing such samples, researchers should first optimize protein extraction protocols to maximize yield from limited patient material. Use multiple TARS2 antibodies targeting different epitopes to ensure detection of potential truncated or variant proteins. Increased sample loading (75-100 μg protein) and extended exposure times may be necessary for Western blots. Consider using more sensitive detection methods such as capillary Western or single-molecule array (Simoa) when conventional Western blotting yields weak signals. Complementary approaches include quantitative PCR to measure transcript levels and cDNA sequencing to identify aberrant splice products, providing context for protein detection results .

What controls should be implemented when using TARS2 antibodies for immunofluorescence studies of mitochondrial localization?

Implementing robust controls for TARS2 immunofluorescence studies is essential for reliable data interpretation. First, include co-staining with established mitochondrial markers such as MitoTracker, TOM20, or COX4 to confirm TARS2's mitochondrial localization. Second, use TARS2-deficient cells (siRNA knockdown or patient-derived cells with confirmed mutations) as negative controls. Third, perform peptide competition assays where the TARS2 antibody is pre-incubated with its immunizing peptide to demonstrate specificity. Fourth, include multiple fixation methods (paraformaldehyde versus methanol) as certain epitopes may be masked by specific fixatives. Fifth, test secondary antibody alone to identify non-specific background. Finally, when studying patient samples, compare multiple control samples to account for natural variation in mitochondrial morphology and TARS2 expression levels .

How can researchers optimize immunoprecipitation protocols for TARS2 to investigate its protein interaction network?

Optimizing TARS2 immunoprecipitation requires careful consideration of multiple factors. Begin with lysis buffer selection—mild non-ionic detergents (0.5-1% NP-40 or Triton X-100) preserve most protein-protein interactions, while more stringent conditions (RIPA buffer) may be needed for strongly associated complexes. Cross-linking with formaldehyde (0.1-1%) prior to lysis can capture transient interactions. Pre-clear lysates with protein A/G beads to reduce non-specific binding. For the immunoprecipitation step, compare multiple commercial TARS2 antibodies to identify those with highest efficiency and specificity. Consider using magnetic beads conjugated with TARS2 antibodies for cleaner results. Include appropriate controls: IgG isotype control, input sample (5-10%), and when possible, TARS2-deficient samples. For interactome analysis, combine with mass spectrometry and validate key interactions through reverse immunoprecipitation and proximity ligation assays.

What methodological approaches can resolve contradictory results between TARS2 antibody detection and RNA-based expression data?

Discrepancies between protein detection and RNA expression data for TARS2 require systematic investigation. First, verify antibody specificity through knockout/knockdown validation and peptide competition assays. Second, assess post-transcriptional regulation by measuring TARS2 protein half-life through cycloheximide chase assays and ubiquitination status via immunoprecipitation followed by ubiquitin blotting. Third, employ polysome profiling to determine translation efficiency of TARS2 mRNA. Fourth, investigate potential protein degradation during sample preparation by including protease inhibitors and comparing multiple extraction protocols. Fifth, use absolute quantification methods (AQUA peptides in mass spectrometry) alongside relative quantification by Western blotting. Finally, consider tissue-specific or condition-specific post-translational modifications that might affect antibody epitope recognition . Documenting results in a systematic table comparing RNA levels, protein detection methods, and potential regulatory mechanisms can help identify patterns explaining the discrepancies.

How should researchers interpret changes in TARS2 protein levels in the context of mitochondrial disease?

Interpreting TARS2 protein levels in mitochondrial disease contexts requires careful consideration of multiple factors. First, establish baseline variation in TARS2 expression across relevant tissue types and developmental stages using age-matched controls. For patient samples, quantify TARS2 levels relative to multiple mitochondrial markers (HSP60, VDAC, COX4) to distinguish TARS2-specific changes from general mitochondrial alterations . Correlate protein levels with functional measurements such as tRNA aminoacylation assays, which directly assess TARS2 activity . Consider the genetic context—certain mutations may affect protein stability rather than expression (e.g., missense mutations) while others lead to unstable transcripts (e.g., splicing mutations) . Analyze multiple patients with similar genetic defects to establish patterns, and where possible, perform longitudinal studies to track disease progression. Finally, integrate TARS2 protein data with clinical parameters and other biochemical markers (lactate levels, respiratory chain complex activities) to develop a comprehensive understanding of disease mechanisms.

What quantitative methods are most appropriate for analyzing TARS2 antibody signals in cancer tissue microarrays?

Analysis of TARS2 expression in cancer tissue microarrays requires rigorous quantitative approaches. For immunohistochemistry, implement digital pathology tools to calculate H-scores (intensity × percentage of positive cells) or Allred scores, ensuring standardization across samples. Use automated image analysis software with machine learning capabilities to segment cellular compartments and quantify mitochondrial-specific TARS2 staining. Calculate tumor-to-normal ratios for each patient to normalize for baseline expression differences. For multiplexed immunofluorescence, employ spectral unmixing to distinguish TARS2 signals from autofluorescence and other markers. Conduct receiver operating characteristic (ROC) analysis to determine optimal cutoff values for categorizing TARS2 expression levels . Present data in comprehensive tables showing expression patterns across cancer types, correlating with gene amplification status from complementary genomic analyses. Statistical comparisons should account for multiple testing and include multivariate analyses to control for confounding factors such as tumor grade, stage, and molecular subtypes.

What emerging applications of TARS2 antibodies should researchers be aware of?

Emerging applications of TARS2 antibodies extend beyond conventional protein detection methods. Researchers should consider using TARS2 antibodies for proximity-based protein interaction studies, such as BioID or APEX2 proximity labeling, to map the spatial proteome surrounding TARS2 in mitochondria. Super-resolution microscopy techniques (STORM, PALM) with TARS2 antibodies can reveal previously undetectable organizational details within mitochondrial translation machinery. In clinical research, development of TARS2 autoantibody assays may provide diagnostic biomarkers for certain mitochondrial disorders . For cancer research, TARS2 antibodies could enable detection of mitochondrial stress responses and altered tRNA charging dynamics associated with metabolic reprogramming . Additionally, combining TARS2 antibody-based proximity ligation assays with metabolic flux analysis can link aminoacylation activity to broader metabolic phenotypes. Finally, the potential involvement of TARS2 in mTORC1 signaling suggests applications in studying nutrient sensing and cellular growth regulation in both normal and disease states .

How can TARS2 antibody research contribute to therapeutic development for mitochondrial diseases?

TARS2 antibody research provides multiple avenues toward therapeutic development for mitochondrial diseases. First, high-throughput screening assays using TARS2 antibodies can identify compounds that stabilize mutant TARS2 proteins or enhance their enzymatic activity. Second, patient-derived cellular models with confirmed TARS2 deficiency (validated by antibody-based methods) serve as platforms for testing gene therapy approaches, including AAV-delivered wild-type TARS2 or base editing to correct specific mutations. Third, TARS2 antibodies enable monitoring of therapeutic responses at the protein level, complementing functional assays and clinical measures. Fourth, for intronic mutations affecting splicing, antisense oligonucleotides can be developed and their efficacy assessed by monitoring TARS2 protein recovery . Fifth, the connection between TARS2 and mTORC1 signaling suggests potential for repurposing existing mTOR modulators for certain TARS2-related conditions . Finally, understanding the three-dimensional structure of TARS2 through crystallography or cryo-EM (facilitated by antibody-based purification) can guide rational drug design targeting specific functional domains or mutation-induced structural alterations.