LARS2 Antibody

What is the optimal application range for LARS2 antibodies in laboratory research?

LARS2 antibodies have been validated for multiple applications with varying optimal dilution ranges. For Western Blot analysis, the recommended dilution typically ranges from 1:500 to 1:2000 . Immunohistochemistry applications generally require dilutions between 1:50 and 1:500, while Immunofluorescence protocols typically use dilutions between 1:20 and 1:200 . For Immunoprecipitation, 0.5-4.0 μg of antibody per 1.0-3.0 mg of total protein lysate is generally effective . It's important to note that optimal dilutions are sample-dependent and should be empirically determined for each experimental system.

Which cell lines have been validated for LARS2 antibody detection?

Several human cell lines have been successfully used for LARS2 antibody validation:

When establishing a new experimental system, it's advisable to include one of these validated cell lines as a positive control to ensure antibody functionality .

What is the subcellular localization pattern of LARS2 when detected by immunofluorescence?

LARS2 primarily localizes to mitochondria as expected for a mitochondrial tRNA synthetase. In immunofluorescence studies, LARS2 typically displays a punctate cytoplasmic pattern consistent with mitochondrial distribution . When conducting IF experiments, co-staining with established mitochondrial markers (such as MitoTracker or antibodies against other mitochondrial proteins) is recommended to confirm proper localization. This pattern can be observed in fixed cells using ethanol fixation (-20°C) . For optimal results, use HepG2 cells which have been well-validated for LARS2 immunofluorescence experiments.

How should sample preparation be optimized for LARS2 detection in different applications?

Sample preparation varies by application:

For Western Blot:

• Use 40-100 μg of whole cell protein depending on expression level

• Solubilize in 1× Laemmli buffer and separate by 12% Tris-Glycine-SDS gels

• The expected molecular weight is approximately 102 kDa

For Immunohistochemistry:

• Heat-mediated antigen retrieval is crucial using either:

  • TE buffer (pH 9.0) (preferred method) or

  • Citrate buffer (pH 6.0) as an alternative

• Paraffin-embedded tissue sections have been successfully used with dilutions around 1:200

For Blue-Native PAGE (BN-PAGE) analysis:

• Solubilize mitochondrial pellets (40 μg) in NativePAGE Sample Buffer containing dodecylmaltoside at a detergent/protein ratio of 6:1

• Include 1 mM AEBSF as a protease inhibitor

What controls should be included when using LARS2 antibodies for experimental validation?

A comprehensive control strategy should include:

• Positive Controls: Include validated cell lines (HepG2, Jurkat, MCF-7) known to express LARS2

• Negative Controls:

  • Primary antibody omission control

  • Non-specific IgG control of the same species and concentration

  • Where possible, LARS2 knockdown/knockout samples for specificity validation

• Specificity Controls:

  • Peptide competition assay using the immunizing peptide

  • Secondary antibody-only controls to assess non-specific binding

For immunoprecipitation experiments, include an input control and IgG control to demonstrate specific enrichment of LARS2 .

What are the considerations for selecting between polyclonal and monoclonal LARS2 antibodies?

For applications studying LARS2 in clinical samples or where quantitative comparisons are critical, monoclonal antibodies offer better reproducibility . For detecting low abundance forms or when maximizing detection sensitivity is the priority, polyclonal antibodies may be preferable .

How can LARS2 antibodies be utilized to study mitochondrial tRNA aminoacylation defects in disease models?

LARS2 antibodies can be employed in several advanced methodologies to investigate mitochondrial tRNA aminoacylation:

• RNA Immunoprecipitation (RIP): LARS2 antibodies have been successfully used to immunoprecipitate LARS2-bound tRNAs in MELAS cybrid cell models, allowing analysis of tRNA interactions . The protocol involves:

  • Crosslinking cells with 1% formaldehyde

  • Isolation of mitochondria using specialized buffers (10 mM Tris-HCl, pH 7.4; 600 mM mannitol; 1 mM EGTA; 0.1% BSA; 1 mM PMSF)

  • Immunoprecipitation using anti-FLAG or anti-LARS2 antibodies

  • RT-qPCR quantification of associated tRNAs

• Rescue Assays: LARS2 antibodies can validate expression of LARS2 carboxy-terminal domain constructs in rescue experiments for MELAS mutations, confirming the chaperonic activity of LARS2 towards mutated tRNAs .

What approaches can be used to investigate LARS2 involvement in mitochondrial translation complexes?

To study LARS2's role in mitochondrial translation complexes:

• Blue-Native PAGE (BN-PAGE) Analysis:

  • Isolate mitochondria from cells of interest

  • Solubilize in NativePAGE sample buffer with dodecylmaltoside

  • Separate on gradient gels and perform western blotting with LARS2 antibodies

  • This approach reveals LARS2 association with higher molecular weight complexes

• Co-immunoprecipitation:

  • Use LARS2 antibodies for pulldown experiments followed by mass spectrometry or western blotting

  • This helps identify novel interaction partners within the mitochondrial translation machinery

• Proximity Labeling:

  • Express LARS2 fused to biotin ligase and identify nearby proteins that become biotinylated

  • Validate interactions using LARS2 antibodies

When investigating these complexes, include analysis of OXPHOS subunits to correlate LARS2 function with mitochondrial translation outcomes .

How should researchers address potential non-specific binding when using LARS2 antibodies in challenging tissue samples?

For challenging tissue samples:

• Optimization of Antigen Retrieval:

  • Test both recommended methods - TE buffer (pH 9.0) and citrate buffer (pH 6.0)

  • Optimize retrieval time and temperature for specific tissue types

• Signal Amplification Strategies:

  • For tissues with low LARS2 expression, consider biotin-streptavidin amplification systems

  • Tyramide signal amplification can increase sensitivity while maintaining specificity

• Dual Validation Approach:

  • Use two different LARS2 antibodies recognizing distinct epitopes

  • Concordant results provide stronger evidence of specific detection

• Blocking Optimization:

  • Test different blocking agents (BSA, normal serum, commercial blockers)

  • Extended blocking times (2-16 hours) may reduce background in problematic tissues

• Cross-Adsorption:

  • Pre-adsorb antibodies with tissue lysates from species with low homology to reduce cross-reactivity

How can discrepancies in LARS2 molecular weight observed in western blot be resolved?

The calculated molecular weight of LARS2 is 102 kDa (903 amino acids) , but researchers sometimes observe discrepancies:

When analyzing western blot data, the validated molecular weight for LARS2 is 102 kDa . Always include positive control samples (HepG2, Jurkat, or MCF-7 cells) to establish the correct band position .

What strategies can address weak or absent LARS2 signal in immunohistochemistry experiments?

For optimizing weak IHC signals:

• Antibody Concentration:

  • Test a range of concentrations, starting with 1:50 dilution for weak signals

  • Consider concentrating the antibody if the stock concentration is insufficient

• Antigen Retrieval Optimization:

  • Extend heat-mediated antigen retrieval time to 20-30 minutes

  • Test both recommended buffers: TE buffer (pH 9.0) and citrate buffer (pH 6.0)

• Detection System Enhancement:

  • Switch to more sensitive detection systems (e.g., polymer-based systems)

  • Consider three-step detection versus two-step methods

• Sample Handling:

  • Ensure tissue fixation time was not excessive (over-fixation masks epitopes)

  • Check tissue processing parameters, including dehydration and clearing protocols

• Signal Amplification:

  • Implement tyramide signal amplification for low abundance targets

  • Use biotin-streptavidin systems with proper blocking of endogenous biotin

How should researchers interpret conflicting data between LARS2 protein expression and functional outcomes?

When facing discrepancies between LARS2 protein levels and functional outcomes:

• Post-translational Regulation Assessment:

  • Investigate phosphorylation status using phospho-specific antibodies

  • Assess ubiquitination to determine if protein is marked for degradation

• Localization Analysis:

  • Perform subcellular fractionation to confirm mitochondrial localization

  • Use immunofluorescence to determine if LARS2 localizes properly to mitochondria despite normal expression levels

• Protein-Protein Interaction Evaluation:

  • Conduct co-immunoprecipitation to assess if LARS2 forms appropriate complexes

  • Investigate potential inhibitory protein interactions

• Functional Activity Testing:

  • Directly measure aminoacylation activity in isolated mitochondria

  • Assess association with target tRNAs using RNA immunoprecipitation

• Alternative Splicing Investigation:

  • Use RT-PCR to identify potential splice variants that maintain epitope recognition but lack full functionality

  • Sequence cDNA to identify potential mutations affecting function but not antibody binding

How can LARS2 antibodies be utilized to investigate Perrault syndrome pathophysiology?

LARS2 mutations have been identified in Perrault syndrome, characterized by hearing loss and ovarian abnormalities . Research approaches include:

• Patient-Derived Sample Analysis:

  • Use LARS2 antibodies to assess protein expression in patient fibroblasts

  • Compare subcellular localization of wild-type versus mutant LARS2 protein

  • Evaluate co-localization with mitochondrial markers

• Functional Consequence Assessment:

  • Examine mitochondrial translation efficiency using pulse-chase labeling

  • Correlate LARS2 protein levels with mitochondrial protein synthesis rates

  • Analyze OXPHOS complex assembly using BN-PAGE followed by western blotting

• Disease Modeling:

  • Validate CRISPR/Cas9-engineered cell models carrying patient mutations

  • Quantify LARS2 protein expression and stability in these models

  • Assess rescue by wild-type LARS2 expression

For Perrault syndrome research, tissue-specific effects should be examined, particularly in cochlear and ovarian tissues, where available .

What considerations are important when using LARS2 antibodies in mitochondrial disease research?

When researching mitochondrial diseases:

• Heteroplasmy Analysis Correlation:

  • In MELAS cybrid models, LARS2 expression should be correlated with mutation heteroplasmy levels determined by PCR/RFLP analysis

  • Document heteroplasmy percentages alongside LARS2 protein data

• Tissue-Specific Expression Patterns:

  • Different tissues show varying LARS2 expression levels

  • Use tissue-specific controls when examining patient biopsies

• Energy Substrate Considerations:

  • Culture cells in media with different energy substrates (glucose vs. galactose)

  • Assess LARS2 expression under metabolic stress conditions

• Mitochondrial Dynamics Assessment:

  • Investigate whether LARS2 dysfunction affects mitochondrial network morphology

  • Correlate LARS2 function with mitophagy markers like BNIP3 and BNIP3L/NIX

• Comprehensive OXPHOS Analysis:

  • Include assessment of multiple OXPHOS complex subunits (NDUFA9, SDHA, UQCRC2, COX I, ATP5A)

  • Correlate LARS2 function with specific complex deficiencies

How can researchers employ LARS2 antibodies in studying the therapeutic potential of aminoacyl-tRNA synthetase fragments?

Research has demonstrated that the carboxy-terminal domain of LARS2 has rescuing activity for certain mitochondrial tRNA mutations . For investigating therapeutic potential:

• Domain-Specific Analysis:

  • Use LARS2 antibodies to validate expression of specific domains (e.g., C-terminal domain or β-strand peptides)

  • Confirm subcellular localization of therapeutic fragments

• Rescue Experiment Validation:

  • After transfecting MELAS cybrid cells with LARS2 constructs, use antibodies to confirm expression

  • Correlate expression levels with functional outcomes

• Delivery Method Development:

  • For exogenously administered peptides, develop antibodies against specific fragments

  • Track internalization and mitochondrial targeting efficiency

• Cross-Mutation Efficacy Testing:

  • Examine effectiveness of LARS2 fragments across different mt-tRNA mutations

  • Use antibodies to standardize expression levels when comparing efficacy

Current research indicates that short β-strand regions (β30_31 and β32_33) within the C-terminal domain show promising rescue activity for both MELAS and MERRF mutations .

What emerging techniques can enhance the application of LARS2 antibodies in single-cell analysis?

For single-cell applications:

• Imaging Mass Cytometry:

  • Metal-conjugated LARS2 antibodies allow quantitative single-cell analysis

  • Simultaneously assess multiple mitochondrial proteins with spatial resolution

• Single-Cell Western Blotting:

  • Microfluidic platforms allow protein analysis from individual cells

  • Optimize LARS2 antibody dilutions for reduced sample input

• Proximity Ligation Assays:

  • Detect protein-protein interactions involving LARS2 at single-molecule resolution

  • Particularly valuable for studying tRNA synthetase complexes

• CODEX Multiplexed Imaging:

  • DNA-barcoded antibodies enable highly multiplexed imaging

  • Study LARS2 in the context of multiple mitochondrial markers

• Live-Cell Nanobody Imaging:

  • Develop nanobodies against LARS2 for live-cell applications

  • Track LARS2 dynamics in response to mitochondrial stress

These emerging techniques will help understand cell-to-cell variability in LARS2 expression and function, particularly relevant for heteroplasmic mitochondrial diseases.

What are the optimal storage and handling conditions for maintaining LARS2 antibody activity?

For maximum antibody stability and performance:

When preparing working dilutions, use freshly prepared buffers and add carrier proteins (0.1-0.5% BSA) for dilute solutions to prevent adsorption to tube walls.

What are the key considerations for validating a new lot of LARS2 antibody before experimental use?

When validating a new antibody lot:

• Side-by-Side Testing:

  • Run parallel experiments with previous lot and new lot

  • Use identical conditions and samples for direct comparison

• Validated Control Samples:

  • Test with established positive controls (HepG2, Jurkat, MCF-7 cells)

  • Include negative controls (primary antibody omission, non-specific IgG)

• Multi-Application Validation:

  • Test in all intended applications (WB, IHC, IF, IP)

  • Verify correct pattern and molecular weight across applications

• Critical Parameter Documentation:

  • Record all experimental conditions (dilutions, exposure times, lot numbers)

  • Create a standardized validation protocol for all future lot testing

• Sensitivity Assessment:

  • Prepare a dilution series of positive control lysate

  • Determine limit of detection compared to previous lot

Maintain a validation report with images from each technique for reference and troubleshooting.

This systematic approach ensures experimental continuity and reliability when transitioning to new antibody lots.