ERF3 Antibody

What is ERF3 and what functional roles does it play in translation?

ERF3 (Eukaryotic Release Factor 3) is a critical GTPase component of the eRF1-eRF3-GTP ternary complex that mediates translation termination in response to stop codons UAA, UAG, and UGA. The primary function of ERF3 is facilitating ETF1/ERF1 delivery to stop codons - the eRF1-eRF3-GTP complex binds to a stop codon in the ribosomal A-site, and subsequent GTP hydrolysis by ERF3 induces a conformational change leading to its dissociation, permitting ETF1/ERF1 to fully accommodate in the A-site .

Beyond translation termination, ERF3 functions as a component of the transient SURF complex which recruits UPF1 to stalled ribosomes during nonsense-mediated decay (NMD) of mRNAs containing premature stop codons . Additionally, ERF3 is required for SHFL-mediated translation termination which inhibits programmed ribosomal frameshifting (-1PRF) of mRNA from viruses and cellular genes .

In mammals, two main isoforms exist: eRF3a (also known as GSPT1) and eRF3b. These isoforms have distinct tissue distribution patterns while sharing functional similarities in translation termination processes.

What types of ERF3 antibodies are available and what are their research applications?

Several ERF3 antibody types are available to researchers, each with specific applications and characteristics:

These antibodies have been validated for applications including Western blotting (typically at 1:1000 dilution), immunoprecipitation, and immunofluorescence . Monoclonal antibodies against human translation termination factor eRF3 have been specifically developed for detecting different epitopes, allowing more precise investigation of protein function and localization .

How can researchers distinguish between eRF3a/GSPT1 and eRF3b in experimental systems?

Distinguishing between eRF3 isoforms requires specific experimental approaches:

• Isoform-specific antibodies: Dedicated antibodies like anti-eRF3a (Thermo Fisher PA5-62621) and anti-eRF3b (Thermo Fisher PA5-60824) allow direct discrimination between isoforms .

• Sample preparation considerations:

  • Temperature sensitivity: Both eRF3a and eRF3b detection is optimal when samples are heated at 37°C for 5 minutes rather than the standard 95°C used for most proteins .

  • Sample concentration should range from 0.1-1 mg/ml for optimal detection .

• Control experiments:

  • Overexpression of FLAG-tagged full-length eRF3a, eRF3a-G575N or eRF3b provides positive controls for antibody validation .

  • Comparison between isoform-specific signals using multiple antibodies confirms specificity.

• Expected molecular weight differences: eRF3a/GSPT1 has a molecular weight of approximately 80 kDa, which can help distinguish it from eRF3b on gel electrophoresis .

These approaches have been successfully employed in studies investigating the distinct functions of eRF3 isoforms in translation termination and mRNA decay pathways.

How should researchers select appropriate ERF3 antibodies for specific experimental applications?

Selection of the optimal ERF3 antibody depends on several key considerations:

• Experimental application:

  • For Western blotting: Most ERF3 antibodies are validated at 1:1000 dilution

  • For immunoprecipitation: Select antibodies specifically validated for IP applications

  • For immunofluorescence: Choose antibodies with demonstrated specificity in cellular localization studies

• Target specificity requirements:

  • Isoform specificity: Determine whether discrimination between eRF3a and eRF3b is necessary

  • Species cross-reactivity: Verify compatibility with your experimental model (human, mouse, etc.)

• Technical factors:

  • Epitope location: Consider whether the antibody recognizes regions that might be masked in protein complexes

  • Functional domains: Some antibodies may recognize regions involved in GTP binding or protein interactions

• Validation status:

  • Published literature: Review citations using the antibody in similar experimental contexts

  • Manufacturer validation: Examine data demonstrating specificity and performance

  • Controls: Plan appropriate positive and negative controls

For example, Cell Signaling's eRF3 Antibody #14980 demonstrates reactivity with human, mouse, rat, and monkey samples, making it suitable for cross-species studies . Conversely, some antibodies like Abcam's ab49878 have been specifically validated with human samples .

How can ERF3 antibodies be utilized in studying translation termination mechanisms?

ERF3 antibodies offer powerful tools for investigating translation termination mechanisms through several advanced approaches:

• Co-immunoprecipitation studies of the termination complex:

  • Immunoprecipitation with anti-ERF3 antibodies pulls down interacting proteins like eRF1

  • Analysis by Western blotting or mass spectrometry identifies complex components

  • Comparison of complex composition under different conditions (e.g., GTP vs. GDP state)

• Kinetic studies of termination factor dynamics:

  • Pulse-chase experiments combined with immunoprecipitation track complex assembly/disassembly

  • Antibodies recognizing different eRF3 conformations can detect GTP hydrolysis-dependent states

• Localization of termination factors:

  • Immunofluorescence microscopy with ERF3 antibodies reveals subcellular distribution

  • Co-staining with ribosomal markers or ER markers provides functional context

  • Studies have confirmed endogenous GSPT1/eRF3a localizes primarily to the endoplasmic reticulum, consistent with its role in translation termination

• Analysis of post-translational modifications:

  • Phosphorylation-specific antibodies can detect regulatory modifications

  • Ubiquitination studies track protein turnover and regulation

These approaches have contributed to our understanding of how the eRF1-eRF3-GTP complex binds to stop codons and how GTP hydrolysis by ERF3 induces conformational changes that enable translation termination .

What methodologies can researchers employ to study ERF3's role in nonsense-mediated mRNA decay (NMD)?

ERF3's involvement in nonsense-mediated mRNA decay can be investigated using these methodological approaches:

• SURF complex (SMG1-UPF1-eRF1-eRF3) analysis:

  • Co-immunoprecipitation with anti-eRF3 antibodies followed by Western blotting for UPF1 (Abcam ab109363, 1:1000) and SMG1 (Cell Signaling 4993, 1:50)

  • Mass spectrometry identification of complex components and their stoichiometry

  • Analysis of temporal dynamics using synchronized cell systems

• Small molecule eRF3 degraders approach:

  • Treatment with compounds like CC-885 or CC-90009 that selectively degrade eRF3a

  • Monitoring effects on PTC readthrough in genetic disease models

  • Western blot analysis with anti-eRF3a antibodies to confirm degradation efficacy

• NMD efficiency measurement:

  • Dual luciferase reporters containing premature termination codons

  • Correlation between eRF3 levels (measured by antibody detection) and NMD activity

  • qPCR quantification of PTC-containing transcripts

• Microscopy approaches:

  • Co-localization of eRF3 with P-bodies or stress granules during NMD

  • Live-cell imaging with fluorescently tagged eRF3 combined with antibody validation

These methods have revealed that eRF3 plays a crucial role in recruiting UPF1 to stalled ribosomes in the context of nonsense-mediated decay of mRNAs containing premature stop codons .

How do small molecule ERF3 degraders affect experimental studies using ERF3 antibodies?

Small molecule ERF3 degraders provide powerful tools for studying eRF3 function but require specific considerations when using ERF3 antibodies:

• Degradation verification:

  • Western blot analysis with anti-eRF3a antibodies (Thermo Fisher PA5-62621, 1:100) confirms reduction in protein levels

  • Dose-response curves establish effective concentration ranges

  • Time-course experiments determine degradation kinetics

• Isoform selectivity considerations:

  • Most degraders like CC-885 and CC-90009 show preferential activity toward eRF3a/GSPT1

  • Parallel analysis with eRF3a-specific and eRF3b-specific antibodies confirms selectivity

  • Overexpression of FLAG-tagged eRF3 variants can test degrader resistance of mutant forms

• Impact on associated protein complexes:

  • Co-immunoprecipitation studies before and after degrader treatment

  • Analysis of effects on eRF1 (detected with mouse anti-eRF1, 1:100, Novus Biologicals 4F9H12)

  • Monitoring UPF1 and SMG1 recruitment to ribosomes

• Readthrough measurement methodologies:

  • Genetic disease models containing premature termination codons

  • Protein detection via automated capillary electrophoresis western analysis

  • Correlation between eRF3a levels and PTC readthrough efficiency

Studies have shown that small molecule degraders can be used effectively to investigate eRF3a's role in translation termination and nonsense-mediated mRNA decay, with antibodies providing crucial validation of target engagement and downstream effects .

What protocols are recommended for detecting protein-protein interactions involving ERF3?

Several methodologies are available for investigating ERF3's protein interaction network:

• Co-immunoprecipitation with anti-ERF3 antibodies:

  • Lysis conditions: Non-ionic detergents (0.5-1% NP-40 or Triton X-100) preserve interactions

  • Pre-clearing step: Reduces non-specific binding

  • Primary antibody: 2-5 μg per 500 μg total protein

  • Critical: Sample heating at 37°C rather than 95°C for analysis

• Bimolecular Fluorescence Complementation (BiFC):

  • ERF3 fusions with YFP fragments detect interactions in living cells

  • Nuclear marker proteins (e.g., CFP-fused Ghd7) confirm subcellular localization

  • This approach has successfully demonstrated protein interactions in the nucleus

• Immunoprecipitation with tagged ERF3 variants:

  • FLAG-tagged ERF3 constructs for pull-down experiments

  • Analysis with anti-FLAG antibody (1:100, Sigma F1804) and antibodies against potential interactors

  • Stable transgenic systems expressing tagged ERF3 provide consistent results

• Cross-linking approaches:

  • Formaldehyde or DSP cross-linking preserves transient interactions

  • Sonication-based lysis maintains complex integrity

  • Reverse cross-linking step prior to SDS-PAGE analysis

For example, researchers investigating ERF3 interactions in rice plants generated stable transgenic lines expressing ERF3-FLAG, performed immunoprecipitation with anti-FLAG antibody, and detected interaction partners by immunoblotting with specific antibodies . Similar approaches can be applied to study eRF3's interactions with translation termination factors and NMD components.

What are the optimal western blotting conditions for ERF3 detection?

Optimal western blotting for ERF3 detection requires careful attention to several technical parameters:

• Sample preparation:

  • Critical temperature consideration: Heat samples at 37°C for 5 minutes for eRF3a and eRF3b detection, not the standard 95°C used for most proteins

  • Protein concentration: 0.1-1 mg/ml for optimal results

  • Lysis buffer: RIPA or NP-40 buffer with complete protease inhibitor cocktail

• Gel electrophoresis:

  • Expected molecular weight: ~80 kDa for ERF3/GSPT1

  • 8-10% polyacrylamide gels provide optimal resolution

  • Include molecular weight markers spanning 50-100 kDa range

• Antibody selection and dilution:

  • Primary antibody: 1:1000 dilution is standard for western blotting

  • Secondary antibody: HRP-conjugated anti-rabbit at 1:2000-1:5000

  • Blocking solution: 5% non-fat milk or BSA in TBST

• Detection methods:

  • Enhanced chemiluminescence (ECL) substrates

  • Alternative: Automated capillary electrophoresis western analysis using ProteinSimple WES system

• Essential controls:

  • Positive control: Lysate from cells overexpressing FLAG-tagged ERF3

  • Negative control: siRNA knockdown of ERF3

  • Loading control: Vinculin (1:600) or GAPDH (1:800)

Researchers have found that sample heating temperature is particularly critical for eRF3 detection, with dramatic differences in signal intensity between samples heated at 37°C versus 95°C. This likely relates to temperature-sensitive epitopes recognized by many ERF3 antibodies .

What considerations are important for immunofluorescence studies with ERF3 antibodies?

Successful immunofluorescence detection of ERF3 requires optimization of several parameters:

• Fixation and permeabilization:

  • Paraformaldehyde (4%) for 15-20 minutes typically preserves ERF3 epitopes

  • Gentle permeabilization with 0.1-0.2% Triton X-100 for 5-10 minutes

  • Alternative: Methanol fixation (-20°C, 10 minutes) may better preserve certain epitopes

• Blocking and antibody incubation:

  • Thorough blocking (5% normal serum, 1 hour) reduces background

  • Primary antibody dilution: Typically 1:100 for anti-ERF3 antibodies in IF applications

  • Extended incubation: Overnight at 4°C for optimal signal development

• Visualization and co-localization:

  • Endogenous GSPT1/eRF3a localizes primarily to the endoplasmic reticulum

  • Co-staining with ER markers provides confirmatory evidence

  • DAPI nuclear counterstain for spatial reference

• Controls and validation:

  • Peptide competition controls confirm antibody specificity

  • siRNA knockdown demonstrates signal specificity

  • Pre-immune serum controls for non-specific binding

• Imaging considerations:

  • Confocal microscopy for precise subcellular localization

  • Z-stack acquisition for complete spatial distribution

  • Consistent exposure settings for comparative analyses

Studies employing these approaches have successfully visualized ERF3 subcellular distribution, confirming its ER localization consistent with its role in translation termination . Bimolecular fluorescence complementation (BiFC) experiments have also revealed nuclear localization of ERF3 when interacting with certain protein partners .

What sample preparation protocols maximize immunoprecipitation efficiency with ERF3 antibodies?

Optimal immunoprecipitation of ERF3 and associated complexes requires careful consideration of sample preparation:

• Lysis buffer composition:

  • Non-denaturing buffer: 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1% NP-40 or Triton X-100

  • Protease inhibitor cocktail: Critical for preventing degradation

  • Phosphatase inhibitors: If studying phosphorylation states

  • Optional: 5 mM MgCl₂ to stabilize nucleotide binding

• Cell/tissue processing:

  • Maintain cold temperature (4°C) throughout processing

  • Gentle homogenization preserves complex integrity

  • Fresh samples yield better results than frozen

• Pre-clearing step:

  • Incubate lysate with protein A/G beads (1 hour, 4°C)

  • Reduces non-specific binding in subsequent steps

• Immunoprecipitation procedure:

  • Antibody amount: 2-5 μg per 500 μg total protein

  • Incubation time: Overnight at 4°C with gentle rotation

  • Bead capture: 1-2 hours with protein A/G beads

  • Washing: 4-5 times with decreasing detergent concentration

• Elution considerations:

  • Critical: Elute/heat at 37°C for eRF3a and eRF3b, not 95°C

  • SDS sample buffer for Western blotting

  • Gentler elution methods for mass spectrometry analysis

This methodology has been successfully applied in studies examining ERF3's interactions with translation factors and other proteins. For example, researchers have generated stable transgenic plants expressing ERF3-FLAG, performed immunoprecipitation with anti-FLAG antibody, and successfully detected interactions with partners like WOX11 through immunoblotting with specific antibodies .

What controls are essential when working with ERF3 antibodies in research applications?

Rigorous control experiments are critical for ensuring reliable results with ERF3 antibodies:

• Positive controls:

  • Overexpression systems: Cells transfected with ERF3 expression constructs

  • Tagged constructs: FLAG-tagged ERF3a, ERF3a-G575N, or ERF3b provide reliable positive controls

  • Known positive tissues/cell types with confirmed ERF3 expression

• Negative controls:

  • Genetic depletion: siRNA or shRNA knockdown of ERF3

  • Peptide competition: Pre-incubation of antibody with immunizing peptide

  • Secondary antibody-only: Detects non-specific secondary binding

• Specificity controls:

  • Multiple antibodies: Different antibodies recognizing distinct ERF3 epitopes

  • Isoform comparison: Anti-eRF3a vs. anti-eRF3b antibodies

  • Species-specificity checks: Testing in predicted reactive and non-reactive species

• Loading and technical controls:

  • Housekeeping proteins: GAPDH (1:800, Abcam ab128915) and Vinculin (1:600, R&D Systems MAB6896)

  • Total protein normalization: Stain-free gels or membrane staining

  • Sample processing controls: Process all samples identically

• Application-specific controls:

  • For Western blotting: Molecular weight markers (~80 kDa expected)

  • For immunofluorescence: Pre-immune serum and peptide competition

  • For immunoprecipitation: IgG control and input samples

Implementation of appropriate controls helps distinguish specific from non-specific signals and validates experimental findings. For instance, research examining the effect of small molecule eRF3a degraders effectively employed FLAG-tagged eRF3 constructs as positive controls while using housekeeping proteins as loading controls .

How can researchers use ERF3 antibodies to study premature termination codon readthrough?

ERF3 antibodies are valuable tools for investigating premature termination codon (PTC) readthrough mechanisms:

• Characterizing small molecule degrader effects:

  • Western blot or capillary electrophoresis quantifies eRF3a depletion after compound treatment

  • Anti-eRF3a antibody (1:100, Thermo Fisher PA5-62621) confirms target engagement

  • Parallel detection of readthrough products from PTC-containing reporter genes

• Genetic disease model applications:

  • Antibodies detect restored full-length protein production in diseases with premature stop mutations

  • Comparison between eRF3a levels and readthrough efficiency establishes correlation

  • Potential therapeutic monitoring applications

• Combined treatment approaches:

  • Study synergistic effects between eRF3 degraders and aminoglycosides (e.g., G418)

  • Western blot analysis of multiple proteins provides comprehensive pathway assessment

  • Antibodies against disease-relevant proteins like Dystrophin (1:400) and TPP1 (1:1000) measure functional outcomes

• Mechanistic investigation methodologies:

  • Analysis of eRF1 (mouse anti-eRF1, 1:100) recruitment to ribosomes

  • UPF1 (rabbit anti-UPF1, 1:1000) binding in PTC contexts

  • Correlation between SURF complex integrity and readthrough efficiency

These approaches have been successfully employed to study how small molecule eRF3a degraders affect PTC readthrough in genetic disease models, with antibody-based detection providing crucial data on both target engagement and functional outcomes .

What methodologies can researchers employ to investigate ERF3 in programmed ribosomal frameshifting?

ERF3's role in inhibiting programmed ribosomal frameshifting (-1PRF) can be studied through several antibody-dependent approaches:

• Frameshifting efficiency measurement:

  • Dual luciferase reporters containing viral or cellular frameshift signals

  • Western blot analysis of ERF3 levels correlates with frameshifting suppression

  • Quantitative relationship between ERF3 abundance and frameshift inhibition

• SHFL-mediated translation termination studies:

  • Co-immunoprecipitation detects ERF3-SHFL interactions

  • Sequential immunoprecipitation reveals complex composition at frameshift sites

  • Correlation between complex formation and frameshifting inhibition

• Viral infection models:

  • Monitor ERF3 levels and localization during viral infection

  • Compare frameshifting efficiency with ERF3 recruitment to viral RNA

  • Analysis of viral countermeasures targeting ERF3 function

• Small molecule modulator applications:

  • ERF3 degraders can be used to modulate frameshifting efficiency

  • Western blot confirmation of ERF3 depletion using specific antibodies

  • Dose-dependent effects on viral replication correlating with ERF3 levels

Research has demonstrated that ERF3 is required for SHFL-mediated translation termination which inhibits programmed ribosomal frameshifting of mRNA from viruses and cellular genes . These methodologies allow researchers to investigate this process in detail and potentially identify therapeutic approaches targeting viral frameshifting.

What are the considerations for using ERF3 antibodies in automated capillary electrophoresis western analysis?

Automated capillary electrophoresis western analysis (e.g., ProteinSimple WES) offers advantages for ERF3 detection but requires specific optimization:

• Sample preparation considerations:

  • Critical temperature sensitivity: Heat samples at 37°C (not 95°C) for eRF3a and eRF3b detection

  • Protein concentration: 0.1-1 mg/ml recommended

  • Sample buffer composition may require adjustment compared to traditional Western blotting

• Antibody dilution optimization:

  • Anti-eRF3a: 1:100 (Thermo Fisher PA5-62621)

  • Anti-eRF3b: 1:100 (Thermo Fisher PA5-60824)

  • Anti-FLAG M2: 1:100 (for tagged constructs)

  • Optimization may differ from traditional Western blotting

• Analysis parameters:

  • Detection profile: High dynamic range setting with multiple substrate injections

  • Multiple exposure times capture optimal signal range

  • Expected molecular weight: ~80 kDa

• Controls and normalization:

  • Housekeeping proteins: GAPDH (1:800) or Vinculin (1:600)

  • Consistency in loading concentration is critical

  • Include positive controls (overexpressed ERF3) for system validation

• Data analysis considerations:

  • Use Compass software (ProteinSimple) for quantification

  • Apply consistent analysis parameters across experiments

  • Statistical validation of quantitative differences

Researchers have successfully employed this technique for detecting both endogenous and overexpressed eRF3a and eRF3b proteins, and for monitoring their levels after treatment with compounds like CC-885 or CC-90009 . The reduced sample volume requirement and improved quantification make this approach valuable for detailed studies of ERF3 biology.

How can researchers interpret contradictory results from different ERF3 antibody applications?

When faced with contradictory results using different ERF3 antibodies, researchers should consider these analytical approaches:

• Epitope mapping and antibody characterization:

  • Determine the exact epitopes recognized by each antibody

  • Monoclonal antibodies against different ERF3 regions may detect distinct conformational states

  • Visualization of epitopes in 3D structural models provides insight into accessibility

• Isoform specificity analysis:

  • Verify whether antibodies distinguish between eRF3a/GSPT1 and eRF3b

  • Test against overexpressed isoform-specific constructs

  • Consider tissue-specific expression patterns of different isoforms

• Technical variables assessment:

  • Sample preparation conditions (particularly temperature sensitivity)

  • Buffer composition effects on epitope accessibility

  • Application-specific optimization (WB vs. IP vs. IF)

• Biological interpretation strategies:

  • Consider post-translational modifications affecting epitope recognition

  • Protein complex formation may mask certain epitopes

  • Conformational changes (e.g., GTP vs. GDP-bound states)

• Validation through orthogonal methods:

  • Mass spectrometry confirmation of target identity

  • Genetic approaches (overexpression, knockdown)

  • Multiple antibodies against different epitopes

Researchers characterizing monoclonal antibodies against eRF3 have demonstrated that different antibodies recognize distinct epitopes, explaining variability in detection across applications . When analyzing contradictory results, methodical investigation of these variables can reconcile apparent discrepancies and provide deeper biological insights.

What emerging research areas will benefit from ERF3 antibody applications?

The continued development and application of ERF3 antibodies will advance several frontier research areas:

• Therapeutic development for genetic diseases with premature stop codons:

  • Monitoring eRF3 levels and target engagement during drug development

  • Correlation of eRF3 modulation with functional protein restoration

  • Personalized medicine approaches based on individual response patterns

• Viral translation control mechanisms:

  • Investigation of viral strategies targeting eRF3 to promote frameshifting

  • Development of antivirals targeting eRF3-dependent translation processes

  • Analysis of host-pathogen interactions at the level of translation termination

• Stress response and translation regulation:

  • ERF3's role in stress granule formation and composition

  • Connections between translation termination and cellular stress responses

  • Post-translational modifications of ERF3 under various stress conditions

• Cancer research applications:

  • Altered eRF3 expression in malignant transformation

  • Connections between NMD efficiency and cancer progression

  • Targeting eRF3 for cancer therapeutics development

These advanced research applications will benefit from continued refinement of ERF3 antibody specificity, sensitivity, and application-specific optimization, driving new discoveries in translation regulation and therapeutic development.

What best practices should researchers follow when publishing studies using ERF3 antibodies?

To ensure reproducibility and rigor in ERF3 antibody-based research, investigators should adhere to these best practices:

• Complete antibody reporting:

  • Manufacturer, catalog number, lot number when relevant

  • Host species, clonality (monoclonal/polyclonal), and isotype

  • RRID (Research Resource Identifier) for unambiguous identification

• Comprehensive methodology details:

  • Sample preparation temperature (critical for eRF3: 37°C vs. 95°C)

  • Dilutions used for each application

  • Complete buffer compositions

  • Incubation times and temperatures

• Validation evidence inclusion:

  • Positive and negative control results

  • Specificity demonstration (knockdown, overexpression)

  • Application-specific validation

  • Full, uncropped blot images in supplementary materials

• Isoform clarification:

  • Specify whether targeting eRF3a/GSPT1, eRF3b, or both

  • Include evidence of isoform specificity if claimed

  • Consider tissue-specific expression patterns in result interpretation

• Acknowledgment of limitations:

  • Discuss potential cross-reactivity

  • Address application-specific constraints

  • Note any contradictory results with different antibodies