RARS Antibody

What is RARS and why are antibodies against it important for scientific research?

RARS (also known as ArgRS or RARS1) is an arginyl-tRNA synthetase that belongs to the class-I aminoacyl-tRNA synthetase family. It catalyzes the attachment of arginine to its cognate tRNA during protein synthesis . Beyond its canonical role, RARS forms part of a macromolecular complex and may be involved in generating inflammatory cytokines .

Antibodies targeting RARS are valuable for:

• Studying protein synthesis mechanisms

• Investigating the multi-tRNA synthetase complex (MSC)

• Exploring non-canonical functions in inflammation and disease

• Examining intracellular localization patterns

The dual role of RARS in both housekeeping functions and potential disease processes makes it a significant target for research using specific antibodies .

How do I select the most appropriate RARS antibody for my specific research application?

Selection of an appropriate RARS antibody should be based on several critical factors:

For advanced applications, consider:

• KD values (equilibrium dissociation constant) which indicate binding affinity

• Recombinant antibodies show 1-2 orders of magnitude higher affinity than traditional monoclonals

• Epitope accessibility in the context of the MSC complex

When studying non-canonical functions, select antibodies targeting regions not obscured by protein-protein interactions .

What are the validated applications for commercially available RARS antibodies?

Based on validated data from multiple sources, current RARS antibodies have been successfully employed in the following applications:

Western blot applications typically show a band at approximately 67-75 kDa, corresponding to the predicted molecular weight of RARS .

What methodological approaches can I use to validate a RARS antibody for my experimental conditions?

Validation of RARS antibodies requires a multi-step approach to ensure specificity and reliability:

• Positive control testing:

  • Use cell lines with confirmed RARS expression (HeLa, A431, or HEK293T cells)

  • Include recombinant RARS protein as a molecular weight reference

• Specificity validation:

  • Cross-reactivity assessment using suspension bead assays (Luminex) against other aaRS family members

  • Knockout/knockdown validation using CRISPR-Cas9 or siRNA approaches

• Application-specific validation:

  • For Western blot: Compare band patterns with predicted molecular weight (75 kDa)

  • For IP-MS: Confirm capture of endogenous RARS and associated complex members

  • For IHC: Compare staining patterns with known RARS expression in tissues

• Binding kinetics assessment:

  • Measure binding affinity (KD) using techniques like surface plasmon resonance

  • Evaluate off-rates which are particularly important for immunoprecipitation applications

When validating polyclonal antibodies, lot-to-lot variation should be carefully assessed to ensure consistent results across experiments .

How can I use RARS antibodies to study the multi-tRNA synthetase complex (MSC)?

RARS is a component of the multi-tRNA synthetase complex (MSC), making antibodies valuable tools for studying this macromolecular assembly:

• Co-immunoprecipitation approaches:

  • Use anti-RARS antibodies to pull down the entire MSC and identify interacting partners

  • IP-MS experiments with anti-RARS have successfully co-immunoprecipitated other MSC components

  • NSAF (Normalized Spectral Abundance Factor) values can provide semi-quantitative information about complex composition

• Complex dynamics investigation:

  • Compare NSAF values of different MSC components to study their relative abundance

  • For example, IP with anti-RARS antibodies revealed lower NSAF values for MetRS, suggesting it may be loosely attached to the complex

• Subcellular localization studies:

  • Combine RARS antibodies with antibodies against other MSC components for co-localization studies

  • Investigate potential nuclear or extracellular localization of RARS outside of the MSC context

• Epitope considerations:

  • Select antibodies targeting exposed epitopes not involved in protein-protein interactions within the MSC

  • Antibodies with low NSAF values in IP-MS may be targeting epitopes obscured in the complex

• Non-canonical function studies:

  • Investigate how RARS modulates the secretion of AIMP1 and generation of the inflammatory cytokine EMAP2

What are the differences between polyclonal, monoclonal, and recombinant RARS antibodies?

Understanding the distinctions between antibody types is crucial for experimental design:

For RARS research, recombinant antibodies offer significant advantages:

• Improved reproducibility across experiments

• Higher binding affinity compared to traditional antibodies

• Well-defined binding characteristics

• Elimination of animal immunization ethical concerns

How can I troubleshoot common issues with RARS antibodies in Western blot applications?

Western blot troubleshooting for RARS antibodies requires systematic assessment of multiple variables:

• Unexpected band patterns:

  • RARS typically shows a band at 67-75 kDa

  • ab128956 shows bands at 60-72 kDa, which may reflect different isoforms or post-translational modifications

  • Non-specific bands may appear if stringency conditions are suboptimal

• Methodological optimization:

  • For ab236788: Use at 1/500 dilution with standard secondary antibody conditions

  • For ab128956: Optimal dilution is 1/1000 for cell lysates at 10 μg loading

  • For ab231610: Use at 2 μg/mL concentration

  • For 27344-1-AP: 1:1000-1:6000 dilution range (sample-dependent)

• Cell line considerations:

  • Verified cell lines include HL-60, K562, A549 , A431, SH-SY5Y, 293T, HepG2 , and HeLa

  • Expression levels vary between cell types, affecting optimal antibody concentration

• Buffer optimization:

  • For membrane proteins or complexes, detergent selection is critical

  • Consider native versus denaturing conditions based on experimental needs

  • TE buffer (pH 9.0) may improve antigen retrieval for certain antibodies

If bands appear at unexpected molecular weights, consider:

• Potential proteolytic degradation (add protease inhibitors)

• Post-translational modifications affecting mobility

• Alternative splicing variants of RARS

• Non-specific binding (increase blocking or washing stringency)

What considerations are important when using RARS antibodies for immunohistochemistry?

Successful immunohistochemistry (IHC) with RARS antibodies requires attention to several methodological details:

• Antibody selection and validation:

  • Validated antibodies include ab236788 , ab231610 , and 27344-1-AP

  • Confirm IHC validation for your specific tissue of interest

• Tissue preparation and antigen retrieval:

  • Paraffin-embedded tissues typically require antigen retrieval

  • For 27344-1-AP: TE buffer pH 9.0 is recommended, with citrate buffer pH 6.0 as an alternative

• Antibody concentration optimization:

  • For ab231610: 20 μg/ml is recommended for IHC applications

  • For 27344-1-AP: 1:50-1:500 dilution range (tissue-dependent)

• Detection systems:

  • DAB staining has been successfully used with ab231610

  • Consider amplification systems for low-abundance targets

• Validated tissue samples:

  • Colon cancer tissue has been validated with ab236788

  • Colorectal and prostate cancer tissues with ab231610

  • Heart tissue and breast cancer tissue with 27344-1-AP

• Controls and interpretation:

  • Include tissues with known RARS expression patterns as positive controls

  • Assess cytoplasmic staining patterns, where RARS is predominantly localized

  • Be aware of potential nuclear or other subcellular localization related to non-canonical functions

How can advanced researchers use deep learning and computational approaches in conjunction with RARS antibodies?

Recent advances in computational biology and AI are opening new avenues for antibody research, including for RARS antibodies:

• Deep learning for antibody design:

  • Generative Adversarial Networks (GANs) can generate novel antibody sequences with desirable properties

  • Wasserstein GAN with Gradient Penalty (WGAN+GP) produces antibodies with high humanness and medicine-likeness

  • These approaches could be applied to develop improved RARS-targeting antibodies

• In-silico antibody generation and screening:

  • Large-scale antibody datasets can be used to train models that predict binding to specific antigens

  • ARPA-H has recently funded a $30 million project to develop AI technologies for therapeutic antibody discovery

  • These approaches could accelerate RARS antibody development for specific applications

• Integrated experimental and computational validation:

  • Computational predictions should be followed by experimental validation

  • Independent laboratory testing is essential to confirm in-silico predictions

  • Biophysical attributes like expression levels, monomer content, and thermal stability should be experimentally verified

• Public antibody response analysis:

  • Mining large antibody datasets can reveal molecular features of public antibody responses

  • Similar approaches could identify common features of antibodies targeting RARS

  • Deep learning models can distinguish between antibodies to different targets based on sequence features

This integration of computational approaches with traditional antibody techniques represents the cutting edge of research methodology.

What techniques can be used to study post-translational modifications of RARS using antibodies?

Post-translational modifications (PTMs) of RARS can significantly impact its function and localization. Studying these modifications requires specialized approaches:

• PTM-specific antibody selection:

  • Consider using antibodies that specifically recognize modified forms of RARS

  • When unavailable, use general RARS antibodies combined with PTM detection methods

• 2D gel electrophoresis with immunoblotting:

  • Separate RARS isoforms by isoelectric point and molecular weight

  • Detect with RARS antibodies to identify charge or size shifts indicative of PTMs

  • The predicted isoelectric point of RARS provides a reference for unmodified protein

• Immunoprecipitation followed by PTM-specific detection:

  • Use RARS antibodies for IP, then probe with antibodies against specific PTMs

  • Alternatively, use PTM-specific antibodies for IP, then detect with RARS antibodies

  • MS analysis of immunoprecipitated RARS can identify specific modification sites

• Phosphorylation studies:

  • Treat samples with phosphatases before immunoblotting to confirm phosphorylation

  • Compare RARS migration patterns before and after treatment

  • Use phospho-specific antibodies in combination with RARS antibodies

• Subcellular fractionation combined with immunoblotting:

  • Different cellular compartments may contain differently modified RARS

  • Use RARS antibodies to track localization patterns of modified forms

  • Compare with markers for specific compartments (cytoplasmic, nuclear, etc.)

These approaches can provide insights into how PTMs regulate RARS functions beyond its canonical role in protein synthesis.

How can I use RARS antibodies to investigate its potential role in disease processes?

RARS has been implicated in various pathological conditions, and antibodies are valuable tools for investigating these connections:

• Cancer research applications:

  • RARS antibodies have been validated in cancer tissues including colon, colorectal, prostate, and breast cancer

  • Compare RARS expression levels between normal and cancerous tissues

  • Investigate potential overexpression patterns, similar to what has been observed with MetRS in non-small cell lung cancer

• Inflammatory disease investigations:

  • RARS may be involved in generating inflammatory cytokines from AIMP1/EMAP2

  • Use RARS antibodies to study its interaction with AIMP1 and potential role in inflammation

  • Investigate potential autoantibody development against RARS in autoimmune conditions

• Non-canonical function studies:

  • Explore RARS secretion in different cell types using antibodies for detection

  • Investigate potential extracellular signaling roles, similar to other aaRS family members

  • Study potential nuclear localization and function using fractionation and immunofluorescence

• Protein-protein interaction networks:

  • Use IP-MS with RARS antibodies to identify novel interaction partners in disease states

  • Compare interaction networks between normal and pathological conditions

  • Investigate dynamic changes in the MSC complex composition

• Therapeutic potential assessment:

  • RARS antibodies can help evaluate it as a potential therapeutic target

  • Study effects of RARS inhibition or modulation on disease-relevant pathways

  • Assess RARS accessibility in different cellular contexts relevant to disease

What are the key considerations for immunofluorescence studies using RARS antibodies?

Immunofluorescence (IF) studies with RARS antibodies require careful attention to several methodological details:

• Antibody selection and validation:

  • Validated antibodies for IF include ab236788

  • Confirm primary antibody compatibility with selected fixation methods

• Fixation and permeabilization optimization:

  • RARS is primarily cytoplasmic, requiring appropriate permeabilization

  • Compare paraformaldehyde fixation with methanol fixation to determine optimal epitope preservation

  • For membrane permeabilization, test Triton X-100, saponin, or digitonin at different concentrations

• Subcellular localization analysis:

  • RARS typically shows cytoplasmic distribution

  • Co-staining with organelle markers can reveal specific localization patterns

  • Z-stack confocal microscopy provides three-dimensional localization information

• Controls and specificity verification:

  • Include secondary-only controls to assess background fluorescence

  • Use siRNA knockdown or CRISPR knockout cells as negative controls

  • Compare staining patterns with multiple RARS antibodies targeting different epitopes

• Co-localization studies:

  • Combine RARS antibodies with antibodies against other MSC components

  • Quantify co-localization using correlation coefficients (Pearson's, Mander's)

  • Investigate dynamic changes in localization under different cellular conditions

• Image acquisition and analysis parameters:

  • Use consistent exposure settings between experimental conditions

  • Apply appropriate background subtraction and thresholding methods

  • Quantify signal intensity and distribution using specialized software

These methodological considerations can significantly improve the reliability and interpretability of IF data for RARS localization studies.

How can I assess cross-reactivity and specificity of RARS antibodies?

Ensuring antibody specificity is critical for reliable experimental results. For RARS antibodies, consider the following approaches:

• Cross-reactivity testing with related proteins:

  • Test against other class-I aminoacyl-tRNA synthetases (LeuRS, ValRS, IleRS)

  • Suspension bead assays (Luminex) can test reactivity against multiple antigens simultaneously

  • Immunoprecipitation followed by mass spectrometry can identify non-specific interactions

• Epitope mapping and sequence analysis:

  • Analyze the immunogen sequence used to generate the antibody

  • Compare with sequences of related proteins to identify potential cross-reactive regions

  • For recombinant protein immunogens, know the specific region (e.g., aa 200-500 for ab236788 or aa 1-150 for ab231610 )

• Validation in knockout/knockdown systems:

  • Use CRISPR-Cas9 knockout or siRNA knockdown of RARS

  • Absence of signal confirms specificity of the antibody

  • Partial reduction in signal with knockdown provides quantitative validation

• Multiple antibody comparison:

  • Use different antibodies targeting distinct epitopes of RARS

  • Concordant results increase confidence in specificity

  • Discrepancies may indicate epitope-specific effects or cross-reactivity issues

• Preabsorption controls:

  • Preincubate antibody with excess recombinant RARS protein

  • Elimination of signal confirms specific binding

  • Persistent signal suggests non-specific interactions

• Testing in diverse cell types:

  • Verify consistent banding patterns across cell lines with known RARS expression

  • Validated cell lines include HeLa, A431, U-251, HEK293T, and others

What emerging technologies are being developed for studying RARS and other aminoacyl-tRNA synthetases?

Recent technological advances are transforming research on RARS and other aaRSs:

• Deep learning-based antibody design:

  • AI approaches can generate novel antibody sequences with optimized properties

  • These technologies could produce highly specific RARS antibodies with improved performance

  • Experimental validation confirms in-silico designed antibodies exhibit high expression, thermal stability, and specificity

• Recombinant antibody production:

  • Moving beyond hybridoma technology to recombinant methods improves consistency

  • Recombinant RARS antibodies show 1-2 orders of magnitude higher affinity

  • Sequence-defined antibodies enable precise epitope targeting and engineering

• Proximity-dependent labeling techniques:

  • BioID or APEX2 fusion with RARS can identify transient interaction partners

  • These methods complement traditional co-immunoprecipitation approaches

  • Can reveal interactions within the MSC not captured by standard antibody techniques

• Structural biology integration:

  • Antibodies can be used as crystallization chaperones for structural studies

  • Single-particle cryo-EM with antibody labeling can reveal complex architectures

  • These approaches could illuminate the structure of RARS within the MSC

• Single-cell analysis technologies:

  • Combining RARS antibodies with single-cell protein profiling methods

  • Reveals cell-to-cell variability in RARS expression and localization

  • May identify rare cell populations with altered RARS function

• CRISPR screening combined with antibody-based readouts:

  • Genome-wide screens to identify regulators of RARS expression or localization

  • Antibody-based detection provides quantitative readouts for screening hits

  • Can uncover novel pathways regulating RARS function

These emerging technologies are expanding our ability to study RARS biology and develop improved research tools.

How should researchers interpret differences in RARS detection across various experimental platforms?

Researchers often encounter variability in RARS detection across different experimental methods. Understanding these differences is critical for accurate data interpretation:

• Molecular weight variations:

  • Western blot detection: Expected at 75 kDa (predicted) , but observed at 60-72 kDa or 67 kDa

  • Variations may reflect:

    • Post-translational modifications

    • Alternative splicing (two cytoplasmic forms differ by 73 amino acids)

    • Proteolytic processing during sample preparation

• Signal intensity differences between applications:

  • Western blot may show strong signals while IF appears weak (or vice versa)

  • Contributing factors include:

    • Epitope accessibility in different sample preparations

    • Protein denaturation affecting epitope recognition

    • Fixation-induced epitope masking or retrieval efficiency

• Subcellular localization discrepancies:

  • RARS is primarily cytoplasmic but may appear in different compartments

  • Consider that:

    • Different fixation methods can affect apparent localization

    • Non-canonical functions may involve translocation to other compartments

    • Some antibodies may detect specific RARS subpopulations

• Complex formation effects:

  • RARS detection may be affected by its incorporation into the MSC

  • Epitopes may be masked by protein-protein interactions

  • Detergent conditions in sample preparation can disrupt or preserve complexes

• Cell type-specific considerations:

  • Expression levels vary between cell types

  • Additional bands may represent cell type-specific isoforms

  • Post-translational modification patterns differ between tissues