QARS Antibody

What is QARS and what is its biological significance?

QARS (glutaminyl-tRNA synthetase) is an enzyme that catalyzes the aminoacylation of tRNA by glutamine. This protein plays essential roles in brain development and the regulation of apoptosis among other biological functions . The human version of QARS has a canonical amino acid length of 775 residues and a protein mass of 87.8 kilodaltons, with 2 identified isoforms . It is predominantly localized in the cytoplasm and is widely expressed across numerous tissue types.

Aminoacyl-tRNA synthetases, including QARS, are fundamental enzymes that link amino acids with their corresponding nucleotide triplets in tRNAs, making them evolutionarily ancient proteins . In metazoans, QARS is one of 9 aminoacyl-tRNA synthetases associated within a multienzyme complex . Mutations in QARS have been linked to progressive microcephaly, highlighting its critical role in neurological development .

What applications are QARS antibodies typically used for in scientific research?

QARS antibodies are utilized across multiple experimental techniques, with the following applications being most common:

These antibodies enable researchers to detect and measure QARS antigen in biological samples, study protein-protein interactions, and investigate the role of QARS in various cellular processes and disease states .

How do I select the appropriate QARS antibody for my experimental needs?

Selection of the optimal QARS antibody should be based on:

• Target specificity: Determine which region of QARS your research focuses on. Some antibodies target specific amino acid sequences, such as AA 1-250, 1-775, 317-569, or 677-775 .

• Host species and clonality: QARS antibodies are available as mouse monoclonal (e.g., clone 5F5) or rabbit polyclonal variants . Monoclonal antibodies offer higher specificity for particular epitopes, while polyclonal antibodies provide broader recognition.

• Validated applications: Verify that the antibody has been validated for your specific application. For example, if conducting immunohistochemistry on paraffin-embedded sections, ensure the antibody is validated for this purpose .

• Species reactivity: Confirm that the antibody recognizes QARS in your experimental model system. Available antibodies show reactivity against human QARS, with some cross-reacting with mouse and rat proteins .

• Antibody form and conjugation: Consider whether you need unconjugated antibodies or those conjugated with specific tags based on your detection system .

What are the recommended storage and handling procedures for QARS antibodies?

For optimal performance and longevity of QARS antibodies:

• Store at -20°C as recommended by manufacturers .

• Avoid repeated freeze-thaw cycles by aliquoting the antibody upon receipt .

• Most QARS antibodies are supplied in buffered aqueous solutions or buffered aqueous glycerol solutions .

• Reconstitute lyophilized antibodies in sterile distilled water with 50% glycerol .

• When shipped in dry ice or wet ice, ensure proper transfer to storage conditions upon receipt .

• Follow manufacturer-specific recommendations for thawing and handling protocols.

What controls should be included when working with QARS antibodies?

To ensure experimental validity and interpretable results:

• Positive controls: Use cell lines known to express QARS, such as HeLa or transfected 293T cells expressing QARS .

• Negative controls: Include samples where QARS expression is absent or significantly reduced.

• Isotype controls: Include an irrelevant antibody of the same isotype (e.g., IgG2bκ for monoclonal antibodies) to assess non-specific binding .

• Epitope competition: Pre-incubate the antibody with the immunizing peptide to demonstrate binding specificity.

• Multiplexed validation: When possible, validate findings with a second QARS antibody targeting a different epitope region.

How can QARS antibodies be employed to investigate mutations associated with neurological disorders?

QARS mutations have been identified as causative factors in progressive microcephaly . When investigating these mutations:

• Mutation-specific approaches: Design experiments that compare wild-type and mutant QARS proteins. Research has demonstrated that mutations such as p.Gly45Val, p.Tyr57His, p.Arg403Trp, and p.Arg515Trp significantly impair QARS aminoacylation activity .

• Structural and functional analysis: Use QARS antibodies to assess how mutations affect:

  • Protein localization through immunofluorescence

  • Protein-protein interactions via co-immunoprecipitation

  • Protein stability through western blotting

• Domain-specific antibodies: Select antibodies targeting specific domains depending on the mutation location. For example, mutations p.Gly45Val and p.Tyr57His are located in the N-terminal domain required for interaction with the multisynthetase complex, while p.Arg403Trp and p.Arg515Trp are in the catalytic core .

• Methodology example: In previous studies investigating QARS mutations, researchers used site-directed mutagenesis to introduce specific point mutations (c.134G>T, c.1207C>T, c.169T>C, and c.1543C>T) into expression vectors. After transfection into cell lines like Neuro2a or Cos7, various antibodies including anti-QARS (SAB1406358, Sigma) were employed for immunoblotting, co-immunoprecipitation, and immunostaining .

What methodologies can be used to study QARS protein-protein interactions within the multisynthetase complex?

For investigating QARS interactions within the multienzyme complex:

• Co-immunoprecipitation protocol:

  • Express recombinant Myc-FLAG-QARS proteins in HEK293T cells

  • Extract proteins using IP buffer (25 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% NP-40, 5% glycerol with protease and phosphatase inhibitors)

  • Clear cell lysates by centrifugation at 16,000 × g

  • Use antibodies against interaction partners, such as anti-RARS (arginyl-tRNA synthetase)

• Antibody selection: For interaction studies, choose antibodies with minimal interference with binding sites. Research has shown that mutations like p.Arg403Trp can disrupt QARS-RARS interaction .

• Protein solubility assessment: Include analysis of protein solubility as mutations such as p.Arg403Trp and p.Arg515Trp have been shown to render QARS less soluble, affecting complex formation .

• Visualization techniques: Combine co-IP with advanced microscopy to visualize complex formation in cellular contexts.

How can antibody-based approaches be optimized for detecting subtle conformational changes in QARS?

For detection of structural dynamics and conformational states:

• Kinetically controlled proteolysis: This emerging technique uses proteases as structural dynamics-sensitive druggability probes. By applying low-Reynolds number flows to make single or few protease incisions, researchers can identify antibody binding sites that change conformation under different conditions .

• Epitope mapping protocols: Generate systematic libraries of antigens with small sequence alterations (elongations, truncations, amino acid exchanges) to create high-affinity binding antibodies targeted to specific conformational states .

• Structural considerations: When designing experiments, consider that proteins like QARS exist in multiple structural conformations with different exposures of regions that may constitute opportunistic targets for antibodies .

• Antibody engineering: Tailor antibodies to elicit optimal binding interactions toward specific epitope regions through detailed knowledge of both the epitope and paratope sequence to yield the required functionality .

What are the technical considerations for using QARS antibodies in multiplex immunoassays?

When incorporating QARS antibodies into multiplex detection systems:

• Antibody compatibility: Ensure all antibodies in the multiplex panel can function under the same conditions without cross-reactivity.

• Species matching: If using multiple primary antibodies, select those raised in different host species to enable species-specific secondary antibody detection.

• Signal optimization: Determine optimal antibody concentrations for each target to achieve balanced signal intensities across all analytes.

• Cross-reactivity testing: Thoroughly test for cross-reactivity between antibodies and non-target proteins, particularly within the aminoacyl-tRNA synthetase family.

• Validation strategy: Use single-plex assays as controls to validate results obtained in multiplex format.

How can QARS antibodies be utilized in therapeutic development approaches?

The emerging field of antibody-based therapeutics offers potential applications for QARS research:

• Targeted delivery systems: Similar to the approach used for KRAS-specific siRNA delivery, antibodies can be chemically coupled to therapeutic agents for targeted delivery to specific cell types .

• Methodology example: In one study targeting KRAS mutations, researchers:

  • Chemically coupled anti-EGFR antibody to siRNA

  • Tested the complex for antibody binding efficiency and serum stability

  • Evaluated the ability to deliver siRNA to EGFR-expressing cells

  • Performed in vitro efficacy testing through western blotting, viability assays, apoptosis assays, and colony formation assays

  • Examined therapeutic activity in in vivo xenograft mouse tumor models

• Rational antibody design: Using kinetically controlled proteases as structural dynamics-sensitive probes can help develop antibodies against difficult-to-target proteins. This approach has successfully created stimulus-selective monoclonal antibodies against targets previously considered undruggable with antibodies .

• Antibody optimization: By systematically altering antigen sequences through elongations, truncations, and amino acid exchanges, researchers can generate antibodies with improved pharmacological function and affinity profiles .

What are the critical factors affecting QARS antibody sensitivity and specificity in immunoassays?

To optimize detection performance in experimental settings:

• Antibody dilution optimization: Conduct titration experiments to determine optimal working dilutions. Recommended ranges include:

  • Western blot: 0.04-0.4 μg/mL or 1-5 μg/mL

  • Immunohistochemistry: 1:50-1:200

  • ELISA: 1/20000-1/80000

• Sample preparation considerations:

  • For proteins extracted from cells, use RIPA buffer with protease inhibitors

  • For immunoprecipitation, use IP buffer (25 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% NP-40, 5% glycerol) supplemented with protease and phosphatase inhibitors

• Assay-specific parameters:

  • For detection of recombinant GST-tagged QARS in ELISA, sensitivity has been reported at approximately 1 ng/mL as a capture antibody

  • For western blotting, QARS appears at approximately 87.8 kDa, though the immunogen used in antibody production may show different molecular weights (e.g., 36.63 kDa)

• Cross-reactivity assessment: Verify species specificity, particularly when working with models other than human samples, as some antibodies show reactivity with mouse and rat QARS while others are human-specific .

What strategies can resolve inconsistent results when using different QARS antibodies?

When facing discrepancies between experiments using different antibodies:

• Epitope mapping: Determine the specific regions recognized by each antibody. Different antibodies may target various domains of QARS, including:

  • AA 1-250

  • AA 1-775

  • AA 317-569

  • AA 677-775

  • AA 215-264

  • AA 514-775

  • AA 725-775

  • N-terminal region

• Isoform specificity: Verify which QARS isoforms are recognized by each antibody, as human QARS has 2 identified isoforms .

• Validation approach: Implement a multi-antibody validation strategy where possible, using antibodies that target different epitopes to confirm findings.

• Protein modifications: Consider post-translational modifications that might affect antibody binding in specific experimental conditions.

• Documentation: Maintain detailed records of antibody performance under various conditions to identify patterns in inconsistencies.

How should researchers interpret and troubleshoot unexpected QARS antibody staining patterns?

When encountering unexpected results:

• Baseline expectations: QARS is primarily localized in the cytoplasm of cells and is widely expressed across many tissue types . Staining patterns should generally reflect this distribution.

• Potential artifacts assessment:

  • Non-specific binding: Implement appropriate blocking procedures and include isotype controls

  • Background issues: Optimize washing procedures and antibody concentrations

  • Fixation artifacts: Compare different fixation methods for their effect on epitope accessibility

• Verification steps:

  • Perform RNA interference or CRISPR/Cas9 knockdown of QARS to confirm specificity

  • Use alternative detection methods (e.g., if unexpected in immunohistochemistry, verify with western blotting)

  • Test the antibody on samples with known QARS expression levels

• Technical considerations:

  • For formalin-fixed, paraffin-embedded sections, ensure proper antigen retrieval methods are employed

  • For immunofluorescence, verify that the fluorophore is compatible with other stains in multiplex assays

What are the implications of QARS mutations for antibody binding and experimental design?

When studying mutant forms of QARS:

• Epitope accessibility: Mutations can alter protein conformation and epitope exposure. Research has shown that mutations like p.Arg403Trp and p.Arg515Trp completely disrupt QARS aminoacylation activity and affect protein solubility .

• Domain-specific considerations:

  • N-terminal domain mutations (p.Gly45Val and p.Tyr57His) affect interactions with the multisynthetase complex and potentially with glutamine tRNA

  • Catalytic core mutations (p.Arg403Trp and p.Arg515Trp) disrupt aminoacylation activity

• Experimental design strategy:

  • Use multiple antibodies targeting different domains

  • Include wild-type QARS as a positive control

  • Consider the use of epitope-tagged recombinant proteins to distinguish endogenous from mutant QARS

• Functional assays: When studying QARS mutations, pair antibody-based detection with functional assays to correlate structural changes with functional outcomes.

How can researchers validate novel QARS antibodies for specific applications?

For comprehensive validation of new antibodies:

• Specificity validation:

  • Test on samples with genetically manipulated QARS expression (overexpression and knockdown)

  • Perform peptide competition assays with the immunizing antigen

  • Test on cell lines known to express or not express QARS

• Application-specific validation:

  • For western blotting: Verify correct molecular weight (87.8 kDa for full-length QARS)

  • For IHC/IF: Compare with established expression patterns and co-localization with known markers

  • For IP: Confirm pull-down of QARS by mass spectrometry

• Cross-platform verification: Validate findings across multiple techniques (e.g., if positive in western blot, confirm with immunofluorescence)

• Reproducibility testing: Evaluate performance across different batches, sample preparations, and experimental conditions.

• Enhanced validation approaches: Consider orthogonal validation methods as employed by some antibody providers, such as genetic, recombinant expression, independent antibody, and orthogonal methods .

How are QARS antibodies contributing to our understanding of neurodevelopmental disorders?

Recent research has established connections between QARS mutations and progressive microcephaly:

• Mechanistic insights: Studies using QARS antibodies have revealed that mutations in QARS can significantly impair its aminoacylation activity. Specifically, variants p.Gly45Val and p.Tyr57His in the N-terminal domain showed over 10-fold reduction in aminoacylation activity, while p.Arg403Trp and p.Arg515Trp in the catalytic core completely disrupted this activity .

• Animal models: In zebrafish models, homozygous qars mutations were investigated using anti-QARS antibodies to understand the relationship between QARS function and neurological development .

• Cellular studies: QARS antibodies have been employed to examine protein interactions in neural cells, helping to elucidate how QARS contributes to brain development. Antibodies used include those against QARS (SAB1406358, Sigma), FLAG (M2, F3165, Sigma-Aldrich; 2368, Cell Signaling), Sox2 (sc-17320, Santa Cruz), Pax6 (PRB-278P, Covance), and HuC/D (A-21271, Life Technologies) .

• Future research directions: Continuing studies may focus on how QARS mutations affect the function of the multisynthetase complex and potentially disrupt neuronal development through altered tRNA aminoacylation or other non-canonical functions.

What novel methodologies are emerging for QARS antibody development and application?

Innovative approaches in antibody technology with relevance to QARS research include:

• Rational antibody design: Using kinetically controlled proteases as structural dynamics-sensitive druggability probes has emerged as a new platform technology for developing antibodies against previously undruggable targets. This approach creates antigens for potential epitopes identified on native-state, disease-relevant proteins in motion .

• Antibody-mediated delivery systems: Similar to techniques developed for KRAS-siRNA delivery, antibodies can be chemically coupled to therapeutic agents for targeted cellular delivery. This approach has shown promise in overcoming resistance to treatments in cancer models .

• High-affinity sequence optimization (hASO): This systematic interrogation of the epitope area with multiple antibodies generated from altered antigens (including elongations, truncations, and amino acid exchanges) can identify optimal binding profiles .

• Recombinant antibody production: Advancements in recombinant technology allow for more consistent antibody production with reduced batch-to-batch variation, improving experimental reproducibility.

How can QARS antibodies be integrated into multi-omics approaches for systems biology research?

For comprehensive analysis of biological systems:

• Integrated proteomics workflows:

  • Use QARS antibodies for immunoprecipitation followed by mass spectrometry to identify interaction partners

  • Combine with transcriptomics data to correlate protein levels with gene expression patterns

  • Integrate with structural biology approaches to map functional domains

• Spatial omics integration:

  • Apply QARS antibodies in spatial proteomics techniques like imaging mass cytometry

  • Correlate with spatial transcriptomics data to understand tissue-specific expression patterns

• Temporal dynamics studies:

  • Utilize QARS antibodies in time-course experiments to track protein dynamics

  • Integrate with metabolomics data to correlate QARS activity with cellular metabolic states

• Systems-level perturbation analysis:

  • Implement CRISPR screens with QARS antibody readouts to identify genetic interactions

  • Combine with phosphoproteomics to understand signaling network connections

What are the key considerations for using QARS antibodies in high-throughput screening applications?

For adaptation to screening platforms:

• Assay miniaturization:

  • Optimize antibody concentrations for microplate formats

  • Determine minimum sample requirements for reliable detection

  • Evaluate signal-to-background ratios in reduced volumes

• Automation compatibility:

  • Test antibody performance with automated liquid handling systems

  • Assess stability under various storage conditions relevant to automated platforms

  • Validate reproducibility across multiple plates and batches

• Multiplexing potential:

  • Evaluate compatibility with other antibodies for simultaneous detection

  • Determine optimal buffer conditions supporting multiple antibody types

  • Assess cross-reactivity in complex sample types

• Data analysis integration:

  • Establish standard curves and detection limits for quantitative applications

  • Develop quality control metrics for large-scale experiments

  • Implement statistical methods appropriate for high-dimensional data

How might QARS antibodies contribute to the development of precision medicine approaches?

Potential applications in translational research and personalized therapeutics:

• Biomarker development:

  • Evaluate QARS levels or localization as potential indicators of disease states

  • Assess correlation between QARS expression and treatment responses

  • Investigate QARS mutations as predictive markers for neurological disorders

• Therapeutic targeting strategies:

  • Using the antibody-siRNA coupling approach demonstrated with KRAS, QARS-targeted therapies could be developed for specific conditions

  • Antibodies could deliver therapeutic agents specifically to cells expressing abnormal levels of QARS

  • The rational antibody design approach using kinetically controlled proteases could generate high-specificity antibodies against particular QARS conformations

• Patient stratification:

  • QARS antibodies could help identify patient subgroups most likely to benefit from specific treatments

  • Correlating QARS status with clinical outcomes could inform treatment decisions

• Companion diagnostics:

  • Development of standardized QARS antibody-based assays to accompany targeted therapeutics

  • Implementation in clinical decision-making algorithms for neurological disorders associated with QARS mutations