ERF1-1 Antibody

What is eRF1 and why is it important in translation research?

eRF1 (also known as ETF1) is a critical component of the eRF1-eRF3-GTP ternary complex that mediates translation termination in response to stop codons. It is responsible for stop codon recognition and inducing hydrolysis of peptidyl-tRNA . eRF1 is essential for cellular viability, as it enables the release of completed polypeptide chains from ribosomes during protein synthesis. The protein is widely expressed across eukaryotic organisms, highlighting its importance in maintaining fundamental cellular functions .
Methodological consideration: When studying eRF1, researchers should consider its interactions with eRF3 and how these interactions are affected by experimental conditions. Antibodies targeting different domains of eRF1 may yield varying results depending on protein conformation in different complexes.

What applications are suitable for eRF1 antibodies?

Based on available commercial antibodies, eRF1 antibodies have been validated for several applications:

• Western blotting (WB)

• Immunocytochemistry/Immunofluorescence (ICC/IF)

• Immunohistochemistry on paraffin sections (IHC-P)
  Methodological consideration: When performing immunofluorescence, optimal fixation methods typically involve 4% paraformaldehyde with permeabilization using 0.1-0.5% Triton X-100. For Western blotting, researchers should expect to detect eRF1 at approximately 49 kDa.

How can I validate eRF1 antibody specificity?

Several approaches for validating eRF1 antibody specificity include:

• Knockdown/knockout controls: Using siRNA or CRISPR to reduce or eliminate eRF1 expression

• Peptide competition assays: Pre-incubating the antibody with the immunizing peptide

• Cross-validation: Using multiple antibodies targeting different eRF1 epitopes

• Positive controls: Testing the antibody on tissues/cells known to express eRF1 (widely expressed)
  Methodological consideration: For knockout validation, the complete loss of eRF1 is lethal to cells due to its essential nature, so inducible or partial knockdown systems are more practical . Using eRF1 nonsense mutants as in yeast models can provide a unique validation system due to feedback-regulated expression .

What are the critical domains of eRF1 to consider when selecting antibodies?

eRF1 has three distinct domains with specific functions:

• N-domain: Responsible for stop codon recognition

• M-domain: Involved in peptidyl-tRNA hydrolysis

• C-domain: Mediates interactions with eRF3
  Methodological consideration: Antibodies targeting the N-domain may interfere with stop codon recognition, while those targeting the C-domain might disrupt eRF3 interactions. For studying specific functions, consider which domain should remain accessible. Commercial antibodies like ab153731 target the C-terminal region (aa 350 to C-terminus) , which might affect detection of eRF1 when it's engaged in certain protein-protein interactions.

How can I use eRF1 antibodies to study the mechanism of eRF1 degraders like SRI-41315 or NVS1.1?

eRF1 degraders represent a novel approach to promoting translational readthrough of premature stop codons. To study their mechanisms:

• Ribosome association assays: Use sucrose gradient fractionation followed by Western blotting with eRF1 antibodies to detect shifts in eRF1 distribution between ribosomal and non-ribosomal fractions

• Ubiquitination detection: Immunoprecipitate eRF1 using antibodies and probe for ubiquitin to measure ubiquitination levels

• Time-course experiments: Track eRF1 degradation kinetics following drug treatment

• Co-localization studies: Use immunofluorescence to visualize eRF1 accumulation at ribosome collision sites
  Methodological consideration: When studying SRI-41315-induced eRF1 degradation, it's important to supplement in vitro systems with recombinant RNF14, as SRI-41315 specifically induces eRF1 ubiquitylation in a dose-dependent manner that is enhanced by the addition of wild-type RNF14 but not ligase-inactive variants . Additionally, ensure active translation is occurring, as SRI-41315-dependent eRF1 ubiquitylation requires active ribosomes .

What methods can I use to study eRF1 interactions with ribosome quality control machinery?

eRF1 degraders like SRI-41315 and NVS1.1 reveal connections between translation termination and ribosome quality control:

• Co-immunoprecipitation: Use eRF1 antibodies to pull down associated factors like RNF14, RNF25, GCN1, and EDF1

• Proximity labeling techniques: BioID or APEX2 fused to eRF1 to identify nearby proteins during termination

• Ribosome collision assays: Monitor recruitment of collision sensors (EDF1, GCN1) in response to eRF1 manipulation

• Polysome profiling: Analyze the distribution of eRF1 and quality control factors across polysome fractions
  Methodological considerations: When studying the recruitment of ribosome collision sensors, researchers should monitor the selective recruitment of factors like EDF1 to ribosomes in cells treated with eRF1 degraders. This phenomenon occurs at concentrations of SRI-41315 sufficient for eRF1 degradation, supporting a model where stabilization of eRF1 on ribosomes produces stalled termination complexes that cause ribosome collisions .

How can I investigate the role of eRF1 ubiquitination in translation termination?

Recent research has highlighted the importance of eRF1 ubiquitination in regulating translation termination:

• Site-directed mutagenesis: Modify potential ubiquitination sites on eRF1 and assess effects on degradation

• E3 ligase knockouts: Generate RNF14 or RNF25 knockout cell lines to study their role in eRF1 ubiquitination

• In vitro ubiquitination assays: Reconstitute the ubiquitination reaction with purified components

• Mass spectrometry: Identify specific ubiquitination sites on eRF1 after drug treatment
  Methodological considerations: Both RNF14 and RNF25 are non-redundant E3 ubiquitin ligases required for ubiquitinating eRF1 in response to eRF1 degraders. Researchers should note that eRF1 ubiquitination was only detected in cells expressing active RNF14 and RNF25 but not in cells depleted for one of the two E3 ligases or in cells expressing mutated versions of RNF14 or RNF25, demonstrating their essential and non-redundant roles .

What approaches can be used to study eRF1 in the context of nonsense-mediated decay (NMD)?

eRF1 plays a crucial role in NMD pathways through the SURF complex:

• Reporter assays: Use GFP or luciferase reporters with premature termination codons (PTCs)

• Co-immunoprecipitation: Pull down eRF1 and probe for NMD factors like UPF1

• Polysome analysis: Study the association of eRF1 with stalled ribosomes at PTCs

• Proximity-dependent biotinylation: Identify proteins near eRF1 during NMD events
  Methodological considerations: When designing reporter constructs, consider using the native stop codon context of eRF1-1 itself, as demonstrated in studies where researchers used the eRF1-1 stop context and terminator region downstream of a GFP reporter lacking its own stop codon . The NMD sensitivity of such constructs can be tested using UPF1 dominant-negative assays.

How can I use eRF1 antibodies to study the effects of eRF1 mutations on translation termination accuracy?

eRF1 mutations can significantly affect translation termination fidelity:

• Readthrough assays: Measure stop codon readthrough using dual-luciferase reporters

• Mutant eRF1 expression: Express mutant forms of eRF1 and assess their localization and function

• Ribosome binding studies: Compare wild-type and mutant eRF1 binding to ribosomes

• Structural analysis: Use cryo-EM to visualize mutant eRF1 interactions with ribosomes
  Methodological considerations: When studying eRF1 mutations, researchers should note that S. cerevisiae eRF1 mutants display significant variability in their stop codon read-through phenotypes depending on the background genotype of the strain used. Evolutionary conservation of amino acids in eRF1 is only a poor indicator of the functional importance of individual residues in translation termination, suggesting complex molecular functions beyond translation termination have shaped eRF1's evolutionary history .

How can I investigate eRF1 misrecognition of sense codons?

Recent research suggests eRF1 can occasionally misrecognize sense codons like UUA:

• In vitro translation assays: Use reporter mRNAs with specific codon usage patterns

• Ribosome profiling: Identify ribosome stalling at specific codons

• Translation rate measurements: Compare elongation rates at different codons

• Competition assays: Test how eRF1 competes with tRNAs at near-cognate codons
  Methodological considerations: When investigating eRF1 misrecognition of sense codons, use reporter mRNAs that predominantly use specific codons of interest. For example, researchers have used nanoluciferase (Nluc) reporters where 11 out of 16 leucine codons were substituted with either CUG (control) or UUA (test) codons. Upon addition of recombinant eRF1-eRF3 to the reaction mixture, decreased protein synthesis was observed specifically from the UUA-rich reporter but not from the CUG-rich reporter, suggesting misrecognition by eRF1 of UUA codons may compete with Leu-tRNA and delay protein synthesis .