EPRS Antibody

What is EPRS and why is it important in research?

EPRS (Glutamyl-Prolyl-tRNA Synthetase) is a bifunctional aminoacyl tRNA synthetase that catalyzes the attachment of glutamate and proline to their cognate tRNAs. This 171kDa protein (UniProt ID: P07814) is also known by several alternative names including "Bifunctional glutamate/proline tRNA ligase" and "Glutamyl prolyl tRNA synthetase" . EPRS is critical in protein synthesis and has been implicated in various cellular regulatory processes beyond its canonical role in translation. Research on EPRS is particularly valuable for understanding fundamental cellular mechanisms and potential implications in disease states.

Which applications are most suitable for EPRS antibody detection?

EPRS antibodies are commonly used in Western Blot (WB) and Immunofluorescence/Immunocytochemistry (IF/ICC) applications . The choice of application should be guided by the antibody's validated reactivity. For instance, antibodies raised against synthetic peptides typically recognize linear epitopes and work well in WB analyses but may not be optimal for detecting native proteins in flow cytometry (FCM), ELISA, or immunoprecipitation (IP) . Conversely, antibodies raised through cDNA or cell immunization often work better with native protein applications but may underperform in WB. Always verify that the specific EPRS antibody has been validated for your intended application.

How should I determine the appropriate dilution for EPRS antibody experiments?

The optimal dilution for EPRS antibodies varies by application, antibody concentration, and specific experimental conditions. While manufacturer recommendations provide a starting point, optimization through titration experiments is essential. For Western blots, begin with a mid-range dilution (e.g., 1:1000) and test a series spanning 1:500 to 1:5000. For immunofluorescence, start with 1:100-1:500. Prepare a dilution series and evaluate signal-to-noise ratio across multiple concentrations. Importantly, antibody dilution optimization should be performed with appropriate positive and negative controls to ensure specificity at the chosen dilution.

What controls should I include when using EPRS antibodies?

For rigorous EPRS antibody experiments, include:

• Positive control: Samples known to express EPRS (e.g., specific cell lines with confirmed EPRS expression)

• Negative control: Samples without EPRS expression or with EPRS knockout/knockdown

• Secondary antibody-only control: Omit primary antibody to detect non-specific binding

• Isotype control: Use matched isotype antibody without specificity for EPRS

• Blocking peptide control: Pre-incubate antibody with immunizing peptide to confirm epitope specificity

These controls help distinguish genuine EPRS signals from background or non-specific binding and are essential for publication-quality data.

Why might I observe different molecular weight bands when using EPRS antibodies?

EPRS is a large protein (171kDa) that may exhibit multiple bands in Western blots due to:

• Alternative splicing variants

• Post-translational modifications

• Proteolytic cleavage products

• Cross-reactivity with related proteins

• Sample preparation artifacts (degradation)

To interpret multiple bands correctly, compare your results with information about known EPRS variants in databases like UniProt . Validation experiments such as knockdown/knockout controls can help confirm which bands represent specific EPRS detection versus non-specific binding. Additionally, denaturing conditions and sample preparation methods may affect epitope availability and band patterns.

How can I optimize EPRS antibody-based co-immunoprecipitation experiments?

For successful EPRS co-immunoprecipitation:

• Antibody selection: Choose antibodies raised against native proteins rather than peptide antibodies, as they better recognize three-dimensional epitopes .

• Lysis buffer optimization: Use gentle, non-denaturing buffers (e.g., RIPA or NP-40 based) to preserve protein-protein interactions.

• Cross-linking strategy: Consider reversible cross-linking with DSP (dithiobis(succinimidyl propionate)) to stabilize transient interactions.

• Pre-clearing: Reduce non-specific binding by pre-clearing lysates with beads and non-specific IgG.

• Bead selection: Compare protein A/G beads with magnetic beads for optimal pulldown efficiency.

• Elution conditions: Test various elution methods (native, acidic, or reducing) to maximize recovery while preserving interacting partners.

Always validate results with reverse co-IP experiments and include appropriate controls to confirm specificity of detected interactions.

What strategies can improve detection specificity when working with EPRS antibodies in complex tissue samples?

Enhancing EPRS detection specificity in complex tissues requires:

• Antigen retrieval optimization: Test multiple methods (heat-induced with citrate or EDTA buffers at varying pH values, or enzymatic retrieval) to maximize epitope accessibility.

• Dual labeling approach: Combine EPRS staining with cell-type-specific markers to confirm expected expression patterns.

• Absorption controls: Pre-absorb antibodies with recombinant EPRS protein to confirm staining specificity.

• Signal amplification: For low-abundance detection, evaluate tyramide signal amplification or higher-sensitivity detection systems.

• Comparative antibody validation: Use multiple antibodies targeting different EPRS epitopes to confirm staining patterns.

• Genetic controls: When possible, utilize tissues from EPRS knockout or knockdown models as negative controls.

These approaches collectively enhance confidence in the specificity of EPRS detection in heterogeneous tissue environments.

How should I address potential cross-reactivity between EPRS antibodies and other tRNA synthetases?

Addressing EPRS antibody cross-reactivity requires a multi-faceted approach:

• Sequence alignment analysis: Conduct bioinformatic comparisons between EPRS and related tRNA synthetases to identify regions of homology that might cause cross-reactivity.

• Epitope mapping: If antibody epitope information is available, evaluate its uniqueness to EPRS versus related proteins.

• Absorption experiments: Pre-incubate EPRS antibodies with recombinant proteins of related tRNA synthetases to identify and eliminate cross-reactive antibodies.

• Validation in knockout/knockdown models: Test antibodies in systems where EPRS is specifically depleted while related synthetases remain expressed.

• Mass spectrometry validation: Confirm the identity of immunoprecipitated proteins by mass spectrometry to detect potential co-precipitation of related synthetases.

This systematic approach helps distinguish between true EPRS signals and potential cross-reactive events with structurally similar proteins.

What considerations are important when studying post-translational modifications of EPRS using antibodies?

Post-translational modification (PTM) studies of EPRS require:

• Modification-specific antibodies: Use antibodies specifically targeting known EPRS PTMs (phosphorylation, acetylation, etc.).

• Preservation of modifications: Optimize sample preparation with appropriate buffers containing phosphatase inhibitors, deacetylase inhibitors, etc.

• Control treatments: Include samples with phosphatase treatment, modification-inducing agents, etc. as controls.

• Enrichment strategies: Consider phospho-enrichment or other PTM-enrichment methods prior to immunoblotting.

• Validation techniques: Confirm antibody-detected modifications using mass spectrometry or other orthogonal methods.

• PTM crosstalk: Evaluate potential interdependence between different modifications on EPRS function.

These approaches enable detailed characterization of how PTMs affect EPRS function and regulation in various biological contexts.

How can I effectively use EPRS antibodies in ChIP-seq experiments to study potential DNA-binding properties?

For successful EPRS ChIP-seq experiments:

• Antibody validation: Verify the antibody's efficiency and specificity in immunoprecipitating EPRS-DNA complexes through pilot ChIP-qPCR experiments.

• Crosslinking optimization: Test various formaldehyde concentrations (0.5-2%) and incubation times to preserve EPRS-DNA interactions without excessive crosslinking.

• Chromatin fragmentation: Optimize sonication conditions to achieve 200-500bp fragments for high-resolution binding site identification.

• Input normalization: Prepare input controls from the same chromatin preparations to accurately normalize enrichment.

• Sequential ChIP: For co-occupancy studies, perform sequential ChIP with EPRS antibodies and antibodies against known DNA-binding partners.

• Spike-in normalization: Include exogenous chromatin (e.g., Drosophila) as spike-in control for quantitative comparisons across conditions.

These methodological considerations ensure robust ChIP-seq data for characterizing potential EPRS-DNA interactions.

What validation steps are essential before using a new EPRS antibody in critical experiments?

Essential validation steps include:

• Literature review: Examine published work using the specific antibody clone/catalog number for applications similar to yours.

• Western blot validation: Confirm single band of expected molecular weight (171kDa for EPRS) in positive control samples .

• Knockout/knockdown validation: Test the antibody in EPRS-depleted samples to confirm specificity.

• Cross-species reactivity: Verify performance across species of interest (human, mouse, etc.) as claimed by manufacturer .

• Peptide competition: Perform blocking experiments with immunizing peptide to confirm epitope specificity.

• Application-specific validation: Test performance specifically in your intended application (WB, IF, IP, etc.).

• Lot-to-lot comparison: For critical experiments, compare performance between antibody lots.

This comprehensive validation approach ensures reliable results and prevents experimental artifacts.

How can I troubleshoot weak or absent signal when using EPRS antibodies?

When encountering weak/absent EPRS antibody signals:

Systematic evaluation of these factors can identify and resolve sensitivity issues in EPRS detection.

What are the most common causes of non-specific binding with EPRS antibodies and how can they be addressed?

Common causes of non-specific binding include:

• Inadequate blocking: Increase blocking time/concentration or try alternative blocking agents (BSA, milk, normal serum).

• Excessive antibody concentration: Titrate primary antibody to find optimal concentration balancing signal and background.

• Cross-reactivity with related proteins: Pre-absorb antibody with recombinant proteins of related family members.

• Secondary antibody issues: Use highly cross-adsorbed secondary antibodies to reduce species cross-reactivity.

• Sample preparation artifacts: Optimize fixation conditions and extraction buffers to reduce non-specific epitope generation.

• Endogenous Ig interference: For tissue sections, use Fab fragments or directly conjugated primaries to avoid endogenous Ig detection.

Addressing these issues systematically can significantly improve signal-to-noise ratio in EPRS detection experiments.

How should I validate EPRS antibody specificity for immunohistochemistry applications?

For IHC validation of EPRS antibodies:

• Positive and negative tissue controls: Test tissues with known EPRS expression patterns and those without expression.

• Genetic models: Utilize EPRS knockout or knockdown tissue sections as definitive negative controls.

• Multiple antibodies: Compare staining patterns with independent antibodies targeting different EPRS epitopes.

• Absorption controls: Pre-incubate antibody with immunizing peptide or recombinant EPRS protein.

• RNA-protein correlation: Compare IHC results with in situ hybridization or RNAseq data from matching tissues.

• Multiple detection methods: Confirm findings using alternate detection systems (e.g., polymer vs. avidin-biotin).

• Isotype controls: Use matched isotype antibodies at identical concentrations to assess non-specific binding.

This multi-parameter validation approach provides confidence in the specificity of EPRS localization in tissue contexts.

What strategies help distinguish between true EPRS signals and artifacts in imaging applications?

To distinguish genuine EPRS signals from artifacts:

• Z-stack analysis: Capture multiple focal planes to confirm signal consistency throughout the cell/structure.

• Co-localization studies: Evaluate co-distribution with known EPRS interaction partners or subcellular markers.

• Signal-depleted controls: Compare with EPRS knockdown samples prepared identically.

• Orthogonal detection methods: Confirm localization patterns with alternative methodologies (e.g., proximity ligation assay).

• Live-cell imaging: When possible, use live-cell compatible EPRS fusion proteins to eliminate fixation artifacts.

• Spectral controls: Include single-label controls to assess and correct for spectral bleed-through in multi-color imaging.

• Super-resolution techniques: Apply methods like STED or STORM to verify subcellular localization beyond diffraction-limited resolution.

These approaches collectively increase confidence in the biological relevance of observed EPRS distribution patterns.

How does the bifunctional nature of EPRS affect antibody selection and experimental design?

EPRS contains discrete glutamyl-tRNA synthetase (ERS) and prolyl-tRNA synthetase (PRS) domains, which impacts experimental approaches:

• Domain-specific antibodies: Select antibodies targeting specific domains based on experimental questions about domain-specific functions.

• Epitope mapping considerations: Determine which domain your antibody recognizes to interpret functional implications correctly.

• Protein interaction studies: Use domain-specific antibodies to distinguish domain-specific protein-protein interactions.

• Conformation-sensitive detection: Consider that inter-domain interactions may mask epitopes in certain functional states.

• Proteolytic processing: Be aware that EPRS can undergo proteolytic processing separating the domains, potentially resulting in multiple bands on Western blots.

• Structural implications: Consider using multiple antibodies against different domains to study conformational changes during EPRS activation or inhibition.

Understanding the bifunctional structure of EPRS is critical for designing experiments that properly distinguish domain-specific functions and interactions.

What methodological adaptations are needed when studying EPRS in different subcellular compartments?

EPRS localizes to multiple subcellular compartments, requiring specific methodological considerations:

• Fractionation protocols: Optimize subcellular fractionation to cleanly separate cytoplasmic, nuclear, and organelle-associated EPRS pools.

• Compartment-specific markers: Always co-stain with established markers (e.g., GAPDH for cytoplasm, lamin for nuclear envelope) to validate fractionation quality.

• Fixation optimization: Different fixatives (PFA vs. methanol) may preferentially preserve EPRS in different compartments.

• Permeabilization conditions: Adjust permeabilization reagents and timing to ensure antibody access to all relevant compartments.

• Specialized imaging: Use confocal or super-resolution microscopy to accurately resolve compartment-specific localization.

• Dynamic studies: Consider live-cell imaging with fluorescently tagged EPRS to track movement between compartments.

These adaptations enable accurate characterization of the dynamic distribution of EPRS across cellular compartments.

How can I determine which epitope an EPRS antibody recognizes and why is this important?

Determining the specific EPRS epitope:

• Manufacturer information: Review detailed product documentation for epitope information .

• Peptide competition: Test a series of overlapping peptides covering the immunogen sequence to identify the minimal recognition sequence.

• Deletion constructs: Express truncated EPRS variants to narrow down the antibody-binding region.

• Domain-specific recognition: Test reactivity against isolated ERS and PRS domains.

• Cross-species analysis: Compare reactivity across species with known sequence variations to identify critical residues.

This information is crucial because:

• Epitope location affects accessibility in different experimental conditions

• Some epitopes may be masked by protein-protein interactions

• Post-translational modifications near the epitope may alter antibody binding

• Epitope knowledge helps predict potential cross-reactivity with related proteins

• Understanding epitope conservation across species informs cross-species applications

What special considerations apply when studying EPRS in the context of the multi-synthetase complex?

EPRS functions within the multi-synthetase complex (MSC), requiring specific experimental approaches:

• Gentle lysis conditions: Use non-denaturing buffers to preserve MSC integrity during extraction.

• Blue native PAGE: Consider native gel electrophoresis to maintain complex integrity for immunoblotting.

• Co-immunoprecipitation strategy: Design pull-down experiments to distinguish between free EPRS and MSC-associated EPRS.

• Sequential immunoprecipitation: Use antibodies against other MSC components followed by EPRS detection to confirm complex association.

• Proximity labeling approaches: Consider BioID or APEX2 fusion proteins to identify proximal proteins in intact cells.

• Stoichiometry analysis: Use quantitative immunoblotting or mass spectrometry to assess EPRS:MSC component ratios.

These approaches help distinguish EPRS functions within versus outside the MSC context.

How do post-translational modifications affect EPRS antibody recognition?

Post-translational modifications can significantly impact EPRS antibody binding:

• Phosphorylation-sensitive epitopes: Phosphorylation near or within the epitope may enhance or inhibit antibody binding.

• Modification-specific antibodies: For studying specific PTMs, use antibodies that specifically recognize modified EPRS.

• Sample treatment effects: Phosphatase or deacetylase treatment may alter antibody recognition patterns.

• Native vs. denatured detection: Some PTM-dependent conformational epitopes may only be detected under native conditions.

• PTM-inducing conditions: Compare antibody reactivity under conditions that promote specific modifications (e.g., kinase activation, stress responses).

• Epitope masking: Consider whether protein-protein interactions induced by PTMs might mask antibody epitopes.

Understanding these interactions between PTMs and antibody recognition is essential for accurately interpreting EPRS detection patterns across different cellular conditions.

How do polyclonal and monoclonal EPRS antibodies compare for different applications?

Comparison of polyclonal vs. monoclonal EPRS antibodies:

Selection should be guided by experimental requirements for specificity, consistency, and application compatibility .

What are the comparative advantages of using EPRS antibodies versus genetic tagging approaches?

Comparison of antibody detection vs. genetic tagging for EPRS studies:

Integrating both approaches provides complementary insights while mitigating the limitations of each method.

What are the best methodological approaches for quantifying EPRS protein levels in different sample types?

Optimal quantification methods by sample type:

For absolute quantification, consider:

• Recombinant EPRS protein standards for calibration curves

• Stable isotope-labeled peptides for mass spectrometry

• Digital PCR for corresponding transcript quantification

• Automated platform analysis for consistency across samples

How do different fixation and permeabilization methods affect EPRS antibody performance in microscopy?

Effects of fixation/permeabilization on EPRS detection:

Optimization recommendations:

• Test multiple fixation/permeabilization combinations

• Consider dual fixation protocols (brief PFA followed by methanol)

• Adjust fixation time and temperature for epitope preservation

• Include antigen retrieval steps if necessary

What are the most effective strategies for multiplex detection of EPRS with other proteins of interest?

Effective multiplex detection strategies:

• Antibody combination planning:

  • Select antibodies from different host species

  • Use directly conjugated primary antibodies with non-overlapping fluorophores

  • Employ isotype-specific secondary antibodies for same-species primaries

• Sequential staining approaches:

  • Apply antibodies sequentially with blocking steps between

  • Consider tyramide signal amplification with antibody stripping between rounds

  • Use specialized multiplexing kits with antibody elution steps

• Advanced imaging techniques:

  • Spectral unmixing for overlapping fluorophores

  • Multi-round imaging with cyclic immunofluorescence

  • Mass cytometry (CyTOF) for high-parameter analysis without fluorescence overlap

• Controls for multiplex validation:

  • Single-stain controls to assess bleed-through

  • Absorption controls to confirm specificity in multiplex context

  • Comparison with individual staining patterns

• Analysis considerations:

  • Use automated colocalization analysis

  • Apply machine learning algorithms for pattern recognition

  • Quantify relative expression levels across multiple markers

These strategies enable simultaneous visualization of EPRS with interaction partners, pathway components, or cellular markers.