EPRS Antibody, HRP conjugated

What is EPRS and why is it significant in research?

EPRS (Glutamyl-prolyl-tRNA synthetase 1) is a bifunctional aminoacyl-tRNA synthetase that catalyzes the attachment of glutamate and proline to their respective tRNAs during protein synthesis. Beyond this canonical role, EPRS has emerged as a critical regulator of cellular signaling pathways. Particularly significant is its recently discovered non-catalytic function in controlling the TGF-β pathway through direct interaction with TGF-β receptor I (TβRI) . This dual functionality makes EPRS an important target in studies of both protein synthesis and fibrosis-related pathologies. EPRS antibodies are essential tools for investigating these various cellular functions through techniques including western blotting, immunohistochemistry, and immunoprecipitation.

What are the structural domains of EPRS and which domains are targeted by common antibodies?

EPRS consists of multiple functional domains that contribute to its various cellular activities:

• EARS1 (Glutamyl-tRNA synthetase domain): Responsible for glutamyl-tRNA synthetase activity

• WHEP domains: Regulatory domains involved in non-canonical functions

• PARS1 (Prolyl-tRNA synthetase domain): Responsible for prolyl-tRNA synthetase activity, further divided into:

  • Anticodon-binding domain (ABD): Mediates interaction with TβRI

  • Catalytic domain: Responsible for enzymatic activity

Research has shown that the PARS1 domain, specifically the anticodon-binding domain (ABD), is responsible for interaction with the glycine-serine rich (GS) domain of TβRI . Commercial antibodies target various epitopes across these domains, with domain-specific antibodies enabling investigation of distinct EPRS functions.

What advantages does HRP conjugation provide for EPRS antibody applications?

HRP (horseradish peroxidase) conjugation offers several methodological advantages in EPRS antibody applications:

• Enhanced sensitivity through enzymatic signal amplification, allowing detection of low-abundance EPRS

• Versatility in detection methods (colorimetric, chemiluminescent, or fluorescent), depending on substrate selection

• Direct detection capability, eliminating the need for secondary antibodies and reducing protocol complexity

• Quantitative analysis potential through measurement of enzymatic activity

• Improved signal-to-noise ratio compared to non-enzymatic detection methods

These advantages make HRP-conjugated EPRS antibodies particularly valuable in techniques requiring high sensitivity, such as western blotting and ELISA for EPRS detection .

How should researchers select the appropriate EPRS antibody for their specific experimental needs?

Selecting the optimal EPRS antibody requires consideration of multiple experimental parameters:

Based on available commercial options, researchers should evaluate antibodies from multiple suppliers to identify those most suitable for their specific applications . Preliminary validation experiments comparing antibody performance in your specific experimental system are strongly recommended.

How can researchers quantify both the binding capacity and functional activity of EPRS antibodies?

Accurate assessment of both antibody loading and functional activity is critical for optimizing EPRS antibody applications. A comprehensive quantification approach includes:

Step 1: Quantify total antibody binding/loading

• For conjugated systems (e.g., antibodies on nanoparticles), quantify unbound antibodies in supernatant using a modified Bradford assay

• Calculate bound antibody by subtracting unbound from total antibody used

Step 2: Assess antibody activity

• For HRP-conjugated antibodies, measure enzymatic activity using appropriate substrates

• For unconjugated antibodies, use antigen-binding assays with excess target antigen

Step 3: Calculate activity ratio

• Determine percentage of active antibodies by comparing actual binding capacity to theoretical binding based on loaded antibody amount

Research has demonstrated significant differences in activity between immobilization methods despite similar loading. For example, studies with anti-HRP antibodies showed that protein A-mediated binding maintained 91±19% activity while direct adsorption resulted in only 23±6% activity, highlighting the importance of measuring both parameters .

What is the relationship between EPRS1 and the TGF-β signaling pathway, and how can antibodies help investigate this interaction?

EPRS1 plays a critical non-catalytic role in TGF-β signaling through a mechanism that can be investigated using specific antibodies:

Co-immunoprecipitation assays using anti-EPRS and anti-TβRI antibodies have been pivotal in demonstrating this interaction. For such experiments, cells are lysed using buffers containing 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 0.5% Triton X-100, 0.5% NP-40, 1 mM EDTA, 1 mM EGTA and protease inhibitors .

How does the method of antibody conjugation affect the activity of EPRS-specific antibodies?

The conjugation method significantly impacts antibody performance and should be carefully considered:

Research has demonstrated that while direct adsorption methods may yield higher antibody loading, oriented immobilization techniques that preserve the antigen-binding regions result in significantly higher percentages of functionally active antibodies . This difference is particularly important when working with expensive or rare antibodies where maximizing functional efficiency is critical.

What methodological approaches can identify and characterize EPRS interactions with binding partners?

Multiple complementary approaches can be employed to characterize EPRS protein interactions:

• Co-immunoprecipitation (Co-IP):

  • Lyse cells in buffer containing 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 0.5% Triton X-100, 0.5% NP-40, 1 mM EDTA, 1 mM EGTA and protease inhibitors

  • Pre-clear lysates with protein A agarose

  • Incubate with anti-EPRS or anti-TβRI antibody overnight at 4°C

  • Add protein A agarose for 2 hours, wash, and analyze by SDS-PAGE

• Pull-down assays for domain mapping:

  • Generate recombinant fragments of EPRS (EARS1, WHEP, PARS1)

  • Perform pull-down assays to identify interaction domains

  • Use microscale thermophoresis (MST) to confirm direct interactions and measure binding affinities

• Subcellular localization studies:

  • Monitor TGF-β-dependent changes in EPRS localization using fractionation

  • Perform immunofluorescence imaging to visualize EPRS translocation to the plasma membrane following TGF-β stimulation

• Functional interaction studies:

  • Use halofuginone (HF) to disrupt EPRS-TβRI interactions

  • Monitor downstream effects on TβRI levels, collagen I, α-SMA, and phospho-SMAD2

  • Assess functional outcomes such as HSC contractility

How can researchers troubleshoot experimental issues with EPRS antibody specificity and sensitivity?

When facing challenges with EPRS antibody performance, consider these methodological approaches:

For specificity issues:

• Validate using multiple antibodies targeting different EPRS epitopes

• Perform peptide competition assays to confirm epitope specificity

• Include proper controls (EPRS knockdown/knockout samples)

• Test antibody cross-reactivity with related aminoacyl-tRNA synthetases

For sensitivity issues:

• Optimize antibody concentration through titration experiments

• Explore signal amplification strategies (tyramide signal amplification, enhanced chemiluminescence)

• Increase sample concentration or antigen retrieval efficiency

• Test different detection substrates for HRP-conjugated antibodies

For inconsistent results:

• Standardize sample preparation protocols

• Consider post-translational modifications that might affect epitope accessibility

• Evaluate buffer compositions and blocking reagents

• Implement quantitative controls in each experiment

What are the optimal experimental conditions for detecting EPRS-TβRI interactions using antibody-based methods?

Based on published research, these conditions optimize detection of EPRS-TβRI interactions:

Cell stimulation conditions:

• TGF-β1 concentration: 2-5 ng/ml

• Stimulation time: 30 minutes to 2 hours for acute interactions

• Cell types: Hepatic stellate cells (HSCs) like LX-2 cells show robust interactions

Co-immunoprecipitation protocol:

• Lysis buffer: 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 0.5% Triton X-100, 0.5% NP-40, 1 mM EDTA, 1 mM EGTA with protease inhibitors

• Pre-clearing: 1 hour with protein A agarose at 4°C

• Antibody incubation: Overnight at 4°C with anti-TβRI antibody

• Bead binding: 2 hours with protein A agarose at 4°C

• Washing: Three times with wash buffer 1 (50 mM Tris-HCl pH 7.4, 150 mM NaCl, 0.5% Triton X-100, 0.5% NP-40) and twice with wash buffer 2 (50 mM Tris-HCl pH 7.4, 150 mM NaCl)

Detection conditions:

• Western blot using anti-EPRS antibody after immunoprecipitation with anti-TβRI

• Reciprocal IP with anti-EPRS followed by anti-TβRI detection confirms interaction

How can researchers use EPRS antibodies to investigate the mechanisms of HF (halofuginone) inhibition?

Halofuginone (HF) exhibits dual inhibitory mechanisms on EPRS function that can be investigated using antibody-based approaches:

Studying catalytic inhibition:

• Measure aminoacylation activity using tRNA charging assays

• Monitor incorporation of radiolabeled amino acids in the presence/absence of HF

Investigating non-catalytic inhibition of EPRS-TβRI interaction:

• Perform in vitro pull-down assays between EPRS and TβRI in the presence/absence of HF

• Use co-immunoprecipitation to assess HF effects on EPRS-TβRI complex formation in cells

• Monitor TβRI protein levels by western blot using anti-TβRI antibodies

• Assess downstream markers (collagen I, α-SMA, phospho-SMAD2) by western blot

• Evaluate functional outcomes such as HSC contractility assays

Research has shown that HF specifically disrupts the EPRS-TβRI interaction, distinguishing it from other tRNA synthetase inhibitors like borrelidin (a TARS1 inhibitor) that do not affect this interaction .

What are the considerations for quantitative analysis of EPRS levels using HRP-conjugated antibodies?

For accurate quantification of EPRS using HRP-conjugated antibodies:

Standard curve development:

• Prepare serial dilutions of recombinant EPRS protein

• Generate standard curves using the same detection system as samples

• Ensure linear response range encompasses expected EPRS concentrations

Signal development optimization:

• Substrate selection: TMB for colorimetric, luminol for chemiluminescence

• Timing: Optimize incubation time to achieve maximum signal while avoiding saturation

• Temperature: Maintain consistent temperature during enzyme reaction

Data analysis approaches:

• For western blots: Use densitometry with appropriate normalization to loading controls

• For ELISA: Apply four-parameter logistic regression for standard curve fitting

• Include quality control samples to monitor inter-assay variability

Potential confounding factors:

• Post-translational modifications may affect antibody recognition

• EPRS complex formation with other proteins might mask epitopes

• Different subcellular pools (MSC-associated vs. free EPRS) may require specific extraction methods

How can EPRS antibodies be used to investigate the role of EPRS in fibrosis development?

EPRS plays a dual role in fibrosis through both its canonical tRNA synthetase activity and non-canonical TGF-β pathway regulation. Research approaches using EPRS antibodies include:

Tissue expression studies:

• Immunohistochemistry with EPRS antibodies to compare expression in normal vs. fibrotic tissues

• Co-localization studies with fibrotic markers (α-SMA, collagen) and TGF-β pathway components

Mechanistic investigations:

• Monitor EPRS phosphorylation status using phospho-specific antibodies following TGF-β stimulation

• Track EPRS dissociation from the multi-tRNA synthetase complex (MSC) using co-immunoprecipitation

• Assess EPRS-TβRI interaction in response to profibrotic stimuli

• Evaluate the effect of EPRS knockdown/inhibition on TGF-β pathway activation

Therapeutic interventions:

• Compare halofuginone analogs for their ability to disrupt EPRS-TβRI interactions

• Develop domain-specific inhibitors targeting the ABD of PARS1 that mediates TβRI binding

• Assess combination approaches targeting both catalytic and non-catalytic EPRS functions

These approaches leverage the mechanistic understanding that EPRS promotes fibrosis by stabilizing TβRI through preventing its ubiquitin-mediated degradation .

What methodological approaches can optimize the orientation and activity of HRP-conjugated EPRS antibodies?

Maximizing functional activity of HRP-conjugated EPRS antibodies requires attention to conjugation strategy:

Research comparing immobilization strategies has demonstrated that while direct adsorption methods may yield higher antibody density, oriented approaches that preserve antigen-binding regions result in significantly higher percentages of functionally active antibodies .

How can researchers distinguish between the catalytic and non-catalytic functions of EPRS using antibodies?

Separating EPRS's dual functions requires strategic experimental approaches:

Domain-specific antibodies:

• Antibodies targeting the catalytic domain of PARS1: Focus on tRNA charging function

• Antibodies targeting the ABD of PARS1: Investigate TβRI interaction

• WHEP domain antibodies: Examine regulatory functions

Functional mutations:

• F1097A and R1152L mutations maintain TβRI interaction despite affecting catalytic activity

• Use antibodies against wild-type and mutant EPRS for comparative studies

Inhibitor-based approaches:

• Halofuginone disrupts both catalytic activity and TβRI interaction

• Compare with catalytic-only inhibitors to differentiate effects

• Use antibodies to monitor differential effects on protein interactions vs. enzymatic activity

Subcellular localization studies:

• Track EPRS translocation to plasma membrane (non-catalytic function) vs. cytoplasmic distribution (catalytic function)

• Use immunofluorescence with EPRS antibodies to visualize TGF-β-dependent translocation

How should researchers interpret conflicting results between different EPRS antibodies?

When facing contradictory results with different EPRS antibodies, follow this systematic approach:

• Epitope mapping analysis:

  • Determine which domains each antibody targets

  • Consider whether post-translational modifications might affect epitope recognition

  • Evaluate whether protein-protein interactions could mask certain epitopes

• Antibody validation assessment:

  • Review validation data for each antibody (western blot, IHC, knockout controls)

  • Test multiple antibodies in parallel on the same samples

  • Include positive and negative controls for each antibody

• Experimental condition evaluation:

  • Determine if differences in sample preparation affect epitope accessibility

  • Consider native vs. denaturing conditions for each application

  • Evaluate fixation methods for microscopy applications

• Biological interpretation:

  • Different antibodies may detect different pools of EPRS (MSC-bound vs. free)

  • Context-dependent interactions might affect epitope accessibility

  • Consider splice variants or proteolytic processing products

• Reconciliation strategies:

  • Use multiple antibodies targeting different epitopes

  • Complement antibody-based methods with non-antibody approaches

  • Report all contradictory findings transparently in publications

What are the potential mechanisms explaining differential efficacy of EPRS antibodies across experimental systems?

Several factors can explain variable antibody performance across systems:

Epitope accessibility variations:

• Conformation changes in EPRS when bound to different partner proteins

• Different extraction methods may preserve or disrupt protein-protein interactions

• Fixation methods for microscopy can differentially affect epitope exposure

Post-translational modifications:

• Phosphorylation status changes following TGF-β stimulation

• Potential glycosylation may affect antibody binding

• Ubiquitination or other modifications in different cellular contexts

Cell type-specific considerations:

• Expression levels of EPRS vary across cell types

• Composition of multi-tRNA synthetase complex differs between tissues

• Relative abundance of binding partners can affect epitope masking

Technical variables:

• Antibody concentration and incubation conditions

• Buffer composition effects on antigen-antibody interactions

• Detection system sensitivity and dynamic range

Understanding these variables helps researchers optimize experimental conditions and interpret seemingly contradictory results in the context of EPRS's complex cellular functions.