NARS Antibody

What is NARS and what are its primary cellular functions?

NARS (Asparaginyl-tRNA synthetase, also known as NARS1 or AsnRS) is an enzyme that catalyzes the attachment of asparagine to tRNA(Asn) through a two-step reaction: first activating asparagine with ATP to form Asn-AMP, then transferring it to the acceptor end of tRNA(Asn) . Beyond its canonical role in protein synthesis, NARS functions as a signaling molecule that induces migration of CCR3-expressing cells . It also plays an essential role in cerebral cortex development, specifically in proper proliferation of radial glial cells . This dual functionality makes NARS antibodies particularly valuable for studying both protein synthesis mechanisms and neurological development processes.

What sample types have validated reactivity with commercial NARS antibodies?

Commercial NARS antibodies have demonstrated confirmed reactivity with human, mouse, and rat samples . Specifically, the rabbit polyclonal NARS antibody (e.g., ab252995) has been validated for use with these species across multiple techniques. When designing experiments, researchers should note that NARS antibodies have been successfully used with various tissue samples, including human rectum, cerebellum, cerebral cortex, and liver tissues, as well as cell lines such as RT4 (human urinary bladder cancer) and U-251 MG (human brain glioma) . Cross-reactivity with other species requires empirical validation before experimental use.

What are the key applications for NARS antibodies in research settings?

NARS antibodies serve multiple experimental purposes across different techniques:

These applications enable researchers to investigate NARS expression patterns, protein interactions, and functional roles in different biological contexts .

How should researchers optimize Western blot protocols for NARS detection?

For optimal Western blot detection of NARS, researchers should consider the following methodological approach:

• Sample preparation: Extract protein using RIPA or NP-40 buffer supplemented with protease inhibitors.

• Gel selection: Use 10% SDS-PAGE gels as NARS has a predicted molecular weight of 63 kDa .

• Transfer conditions: Transfer to PVDF membranes at 100V for 1 hour or 30V overnight for larger proteins.

• Blocking: Block with 5% non-fat milk or BSA in TBST for 1 hour at room temperature.

• Primary antibody incubation: Dilute anti-NARS antibody to 0.4 μg/mL in blocking buffer and incubate overnight at 4°C .

• Detection: Use HRP-conjugated secondary antibodies with appropriate chemiluminescent substrates.

• Expected results: Verify the presence of a band at approximately 63 kDa, confirming NARS detection .

Inclusion of positive controls (e.g., RT4 or U-251 MG cell lysates) helps validate experimental success, while negative controls (secondary antibody only) confirm specificity .

What considerations are important when using NARS antibodies for immunohistochemistry?

When performing immunohistochemistry with NARS antibodies, researchers should implement the following protocol optimizations:

• Tissue fixation: Use 10% neutral buffered formalin for consistent preservation of antigenic epitopes.

• Antigen retrieval: Perform heat-induced epitope retrieval in citrate buffer (pH 6.0) or EDTA buffer (pH 9.0) to unmask antigens.

• Blocking: Use 3% hydrogen peroxide to block endogenous peroxidases, followed by 5-10% normal serum from the species of the secondary antibody.

• Antibody dilution: Based on published results, a 1/5000 dilution of NARS antibody is recommended for paraffin-embedded tissues .

• Incubation conditions: Incubate primary antibody overnight at 4°C in a humidified chamber.

• Counterstaining: Use hematoxylin for nuclear visualization after DAB development.

• Controls: Include cerebellum or cerebral cortex tissues as positive controls given demonstrated reactivity .

These methodological considerations ensure specific staining and reliable interpretation of NARS expression in tissue sections.

How can researchers troubleshoot non-specific binding issues with NARS antibodies?

When encountering non-specific binding with NARS antibodies, implement these methodological solutions:

• Antibody validation: Verify antibody specificity through knockout/knockdown controls or peptide competition assays.

• Blocking optimization: Increase blocking agent concentration to 5-10% and extend blocking time to 2 hours.

• Antibody titration: Perform dilution series tests (1:1000 to 1:10,000) to identify optimal antibody concentration.

• Buffer modifications:

  • Add 0.1-0.5% Triton X-100 to reduce hydrophobic interactions

  • Increase salt concentration (150-500 mM NaCl) to disrupt low-affinity binding

  • Add 0.1-1% BSA as a carrier protein

• Washing stringency: Increase wash steps (5-6 times) and duration (10 minutes each) with gentle agitation.

• Secondary antibody cross-reactivity: Use highly cross-adsorbed secondary antibodies and include serum from the host species.

• Tissue-specific considerations: For brain tissues with high lipid content, extend deparaffinization steps and consider using Sudan Black B to reduce autofluorescence .

Systematic application of these troubleshooting steps will help identify and eliminate sources of non-specific binding.

What quality control measures ensure reliable results when using NARS antibodies?

To ensure experimental reliability with NARS antibodies, implement these quality control measures:

• Antibody validation:

  • Verify lot-to-lot consistency through standard curve analysis

  • Confirm absence of contaminating antibodies through isotype controls

  • Validate purity (>95% recommended) through SDS-PAGE analysis

• Experimental controls:

  • Positive tissue controls (cerebellum, cerebral cortex, liver)

  • Negative controls (omission of primary antibody)

  • Isotype controls (matched non-specific antibody at same concentration)

• Technical validation:

  • Run parallel detection with two different NARS antibodies targeting distinct epitopes

  • Use alternative detection methods (e.g., mass spectrometry) to confirm identified bands

  • Verify cellular localization patterns through fractionation studies

• Data documentation:

  • Record antibody source, catalog number, lot number, and dilution

  • Document exposure times for imaging

  • Maintain standardized protocols across experiments

These quality control measures enhance experimental reproducibility and result interpretation.

How can researchers use NARS antibodies to investigate its dual role in protein synthesis and neurological development?

To investigate NARS's dual functionality, researchers should implement these methodological approaches:

• Differential function analysis:

  • Use site-directed mutagenesis to generate NARS variants with impaired aminoacylation activity but intact signaling capacity

  • Apply these variants in complementation assays to distinguish between functions

  • Utilize domain-specific antibodies to track differential localization

• Developmental studies:

  • Perform time-course analysis of NARS expression in cerebral cortex during development

  • Correlate NARS levels with radial glial cell proliferation markers

  • Use conditional knockout models with temporal control to determine critical developmental windows

• Protein-protein interaction mapping:

  • Conduct co-immunoprecipitation with NARS antibodies followed by mass spectrometry

  • Perform proximity ligation assays to identify in situ interaction partners

  • Use FRET/BRET assays to validate direct protein interactions

• Signaling pathway interrogation:

  • Apply pharmacological inhibitors of CCR3 signaling while monitoring NARS activity

  • Use phospho-specific antibodies to track signaling cascade activation

  • Implement RNA-seq after NARS knockdown to identify downstream effectors

These advanced approaches enable dissection of NARS's distinct roles and their mechanistic underpinnings in different cellular contexts .

What are the optimal approaches for using NARS antibodies in complex tissue systems with multiple cell types?

When investigating NARS in heterogeneous tissue systems, implement these specialized methodological strategies:

• Cell type-specific analysis:

  • Combine NARS immunostaining with cell type-specific markers in multiplexed immunofluorescence

  • Implement laser capture microdissection followed by Western blot to isolate specific cell populations

  • Use flow cytometry with permeabilization for intracellular NARS detection in mixed cell populations

• Spatial distribution analysis:

  • Apply tissue clearing techniques (CLARITY, iDISCO) for whole-organ NARS visualization

  • Implement high-resolution confocal or light-sheet microscopy for 3D reconstructions

  • Correlate NARS expression with tissue architecture through digital spatial profiling

• Single-cell resolution approaches:

  • Combine immunohistochemistry with RNAscope to correlate protein and mRNA levels

  • Perform image cytometry on tissue sections for quantitative single-cell analysis

  • Use CyTOF with metal-conjugated NARS antibodies for high-dimensional phenotyping

• Dynamic regulation studies:

  • Implement intravital microscopy with fluorescently tagged NARS antibodies in animal models

  • Correlate NARS levels with functional readouts through simultaneous electrophysiology recordings

  • Track changes during pathological processes through longitudinal sampling approaches

These advanced techniques enable comprehensive analysis of NARS expression and function across diverse cell types within complex tissue environments .

How might bispecific antibody technologies enhance NARS-targeted research?

Bispecific antibody approaches offer innovative methodological advantages for NARS research:

• Dual targeting strategies:

  • Design bispecific antibodies targeting NARS and its interaction partners simultaneously

  • Create constructs recognizing both NARS and markers of specific subcellular compartments

  • Develop antibodies targeting NARS and tissue-specific antigens for selective visualization

• Advanced therapeutic applications:

  • Engineer bispecific antibodies linking NARS recognition with immune cell recruitment

  • Create constructs targeting both inflammatory responses and NARS signaling

  • Develop antibodies capable of modulating NARS activity through conformation-selective binding

• Technical implementation:

  • Apply phage display technology for rapid screening of bispecific antibody libraries

  • Utilize computational approaches to predict optimal epitope combinations

  • Implement antibody engineering to optimize binding affinity and specificity

The application of bispecific antibody technologies represents a significant advancement in NARS research, enabling simultaneous targeting of multiple disease pathways and improved therapeutic efficacy with reduced side effects .

What role do machine learning algorithms play in advancing NARS antibody development and application?

Machine learning approaches are transforming NARS antibody research through these methodological innovations:

• Epitope prediction and optimization:

  • Apply deep learning algorithms to predict antibody-antigen interactions with increased accuracy

  • Use neural networks to identify optimal epitopes within the NARS protein structure

  • Implement computational models to predict cross-reactivity and minimize off-target binding

• Experimental design enhancement:

  • Utilize predictive algorithms to optimize antibody screening protocols

  • Apply machine learning to identify optimal buffer conditions and reduce non-specific binding

  • Implement automated image analysis for standardized interpretation of immunostaining results

• Data integration and analysis:

  • Develop algorithms that integrate antibody binding data with functional outcomes

  • Create predictive models connecting NARS expression patterns with disease progression

  • Implement deep learning approaches for pattern recognition in complex tissue systems

• Production optimization:

  • Apply machine learning to optimize hybridoma selection and antibody expression

  • Utilize predictive models to enhance stability and reduce batch-to-batch variation

  • Implement computational approaches to predict and mitigate potential immunogenicity

These machine learning applications are dramatically reducing antibody development timelines from years to months while enhancing specificity and efficacy for research applications .