ERF1-2 Antibody

What is the difference between eRF1 isoform 1 and isoform 2?

eRF1 isoform 1 (eRF1iso1) represents the canonical, well-studied form of the eukaryotic release factor involved in translation termination. Isoform 2 (eRF1iso2) is 33 amino acid residues shorter than isoform 1, primarily due to differences in the N-terminal region. While isoform 1 is widely expressed across tissues, isoform 2 has distinct functional properties, including decreased codon recognition and peptide release activities, and exhibits unipotency to UGA stop codons. eRF1iso2 can interact with ribosomal subunits and pre-termination complexes, but stimulates eRF3a GTPase activity significantly less efficiently than isoform 1 .

How do I select the appropriate antibody to specifically detect eRF1 isoform 2?

When selecting antibodies for specific detection of eRF1 isoform 2, consider epitope location carefully. Many commercial antibodies target the C-terminal domain of eRF1, which is present in both isoforms. For isoform-specific detection, choose antibodies targeting unique regions of eRF1iso2 or utilize the size difference between isoforms (eRF1iso2 runs at a lower molecular weight on SDS-PAGE). Western blot analysis can separate these isoforms, as demonstrated in studies where researchers successfully distinguished eRF1iso2 from eRF1iso1 based on molecular weight differences . Additionally, validate antibody specificity using recombinant proteins or cells with known expression patterns of each isoform.

What are the recommended positive control samples for eRF1-2 antibody validation?

For validating eRF1-2 antibodies, optimal positive controls include ribosomes purified from HeLa cells, placenta tissue, rabbit reticulocyte lysate (RRL), and mouse Krebs-2 ascites cells, all of which have demonstrated detectable levels of eRF1iso2 associated with 60S ribosomal subunits . Recombinant eRF1iso2 protein expressed in bacteria can serve as an additional validation control. Western blot analysis should reveal a distinct band at approximately 47 kDa (compared to the 50 kDa band of eRF1iso1). Research has confirmed the expression of eRF1iso2 in human, rabbit, and mouse tissues, indicating conserved expression across mammalian species .

What are the optimal conditions for Western blot detection of eRF1 isoform 2?

For optimal Western blot detection of eRF1 isoform 2, use the following protocol:

• Sample preparation: Extract proteins from cells or tissues using RIPA buffer supplemented with protease inhibitors.

• Electrophoresis: Load 20-30 μg of protein on 10-12% SDS-PAGE gels to achieve adequate separation of eRF1 isoforms.

• Transfer: Use PVDF membranes with a standard wet transfer system (100V for 1 hour or 30V overnight at 4°C).

• Blocking: Block with 5% non-fat milk in TBST for 1 hour at room temperature.

• Primary antibody: Dilute anti-eRF1 antibody 1:1000 in blocking solution and incubate overnight at 4°C .

• Washing: Wash membranes 3× for 5 minutes each with TBST.

• Secondary antibody: Use HRP-conjugated anti-rabbit secondary antibody (1:5000) for 1 hour at room temperature.

• Detection: Visualize using enhanced chemiluminescence (ECL) substrate.

eRF1iso2 should appear at approximately 47 kDa, while eRF1iso1 will appear at approximately 50 kDa . Extended running time may be necessary to clearly separate these closely sized isoforms.

How can I optimize immunocytochemistry protocols for detecting cellular localization of eRF1 isoform 2?

For optimal immunocytochemistry detection of eRF1 isoform 2:

• Cell preparation: Grow cells to ~70% confluence on glass coverslips.

• Fixation: Fix with absolute methanol for 5 minutes at -20°C, followed by post-fixation with 3% paraformaldehyde for 15 minutes at 4°C.

• Permeabilization: Treat with 0.5% Triton X-100 for 5 minutes at room temperature.

• Blocking: Block with 10% FBS containing 0.1% glycine in PBS for 1 hour at room temperature.

• Primary antibody: Incubate with anti-eRF1 antibody (1:100-1:200 dilution) for 1 hour at room temperature or overnight at 4°C.

• Washing: Wash cells with PBS (3× for 5 minutes each).

• Secondary antibody: Incubate with fluorophore-conjugated secondary antibodies (e.g., Alexa Fluor 555 for rabbit IgG) for 1 hour at room temperature.

• Mounting: Mount using media containing DAPI for nuclear counterstaining .

For stress granule co-localization studies, consider co-staining with anti-G3BP1 antibodies as markers. Under certain stress conditions, translation factors may relocalize to distinct cytoplasmic compartments .

What methods can be used to study the interaction between eRF1 isoform 2 and other translation termination factors?

To study interactions between eRF1iso2 and other translation termination factors:

• Co-immunoprecipitation (Co-IP):

  • Use anti-eRF1 antibodies to pull down protein complexes from cell lysates

  • Analyze co-precipitated proteins (such as eRF3a/b) by Western blotting

  • Include RNase treatment to distinguish RNA-mediated from direct protein interactions

• Sucrose gradient fractionation:

  • Separate ribosomal complexes on 10-50% sucrose gradients

  • Collect fractions and analyze by Western blotting for eRF1iso2 and potential binding partners

  • Studies have demonstrated that eRF1iso2 associates primarily with 60S ribosomal subunits

• Proximity-based labeling:

  • Express BioID or TurboID fusions of eRF1iso2 in cells

  • Identify biotinylated proteins using streptavidin pulldown followed by mass spectrometry

  • This approach can identify transient or weak interactions difficult to capture by Co-IP

• Recombinant protein interaction assays:

  • Express and purify recombinant eRF1iso2 and potential binding partners

  • Perform in vitro binding assays such as GST pulldown or surface plasmon resonance

  • Measure binding kinetics and affinities between purified components

Research has shown that while eRF1iso2 can interact with eRF3a, it stimulates GTPase activity significantly less efficiently than eRF1iso1 .

What are common issues when detecting eRF1 isoform 2 by Western blot and how can they be resolved?

Common issues when detecting eRF1iso2 by Western blot include:

• Poor band separation between isoforms:

  • Use longer SDS-PAGE gels (15-20 cm) with 10-12% acrylamide

  • Run at lower voltage (80-90V) for extended periods

  • Consider using Phos-tag gels to enhance separation if phosphorylation differences exist

• Cross-reactivity with eRF1iso1:

  • Pre-absorb antibodies with recombinant eRF1iso1 protein

  • Use antibodies raised against unique regions of eRF1iso2

  • Confirm specificity through knockout/knockdown validation

• Low signal intensity:

  • Enrich for ribosome-associated fractions, as eRF1iso2 appears primarily associated with 60S ribosomal subunits

  • Increase protein loading (30-50 μg)

  • Extend primary antibody incubation time to overnight at 4°C

  • Use signal enhancement systems such as biotin-streptavidin amplification

• Degradation products:

  • Include protease inhibitors in all buffers

  • Keep samples cold throughout preparation

  • Add N-ethylmaleimide to inhibit deubiquitinating enzymes if studying ubiquitinated forms

How can I distinguish between true eRF1 isoform 2 signal and potential antibody artifacts?

To distinguish true eRF1iso2 signal from artifacts:

• Validation controls:

  • Include recombinant eRF1iso1 and eRF1iso2 proteins as size markers

  • Use siRNA knockdown of specific isoforms (targeting unique regions)

  • Include tissue/cell samples known to express different ratios of isoforms

• Multiple antibody approach:

  • Use at least two different antibodies targeting different epitopes of eRF1

  • Compare detection patterns across antibodies

  • Studies have used antibodies against both N- and C-terminal domains to evaluate potential changes in antibody avidity

• Mass spectrometry validation:

  • Excise the band corresponding to eRF1iso2 from gels

  • Perform tryptic digestion and mass spectrometry analysis

  • Confirm the presence of peptides unique to eRF1iso2

• Isoform-specific expression constructs:

  • Express tagged versions of each isoform separately

  • Use as controls to validate antibody specificity and cross-reactivity

When evaluating whether protein level changes are due to actual expression differences versus antibody detection issues, consider post-translational modifications that might affect epitope recognition .

How can I assess the relative contributions of eRF1 isoform 1 versus isoform 2 in translation termination?

To assess the relative contributions of eRF1 isoforms to translation termination:

• Isoform-specific depletion:

  • Design siRNAs or CRISPR guides targeting unique regions of each isoform

  • Measure effects on stop codon readthrough using dual luciferase reporters with different stop codons (UAA, UAG, UGA)

  • Research has shown that eRF1iso2 exhibits unipotency to UGA stop codons

• Reconstituted in vitro translation systems:

  • Set up mammalian in vitro translation systems with defined components

  • Add purified recombinant eRF1iso1 or eRF1iso2 at varying concentrations

  • Measure peptide release activity and stop codon recognition efficiency

  • Studies demonstrate that eRF1iso2 has decreased codon recognition and peptide release activities compared to eRF1iso1

• Ribosome profiling:

  • Perform ribosome profiling in cells with altered ratios of eRF1 isoforms

  • Analyze ribosome density at stop codons and in 3' UTRs

  • Recent techniques like selective Monosome-Seq with SBP-tagged eRF1 can enrich for ribosomes directly bound to eRF1

• GTPase activation assays:

  • Measure the ability of each isoform to stimulate eRF3's GTPase activity

  • Research indicates eRF1iso2 stimulates eRF3a GTPase activity significantly less efficiently than eRF1iso1

How does cellular stress affect the expression and localization of eRF1 isoform 2?

To investigate stress-induced changes in eRF1iso2 expression and localization:

• Stress induction protocols:

  • Treat cells with various stressors (e.g., arsenite, heat shock, ER stress inducers)

  • Monitor changes in eRF1iso2 levels by Western blot

  • Track subcellular localization by immunofluorescence microscopy

• Stress granule association:

  • Co-stain for stress granule markers (G3BP1, eIF3b) and eRF1

  • Perform time-course experiments to track dynamic relocalization

  • Research shows that translation termination factors can relocalize under stress conditions

• Quantitative analysis:

  • Use quantitative PCR to measure transcript levels of different splice variants

  • Perform polysome profiling to assess translation status of eRF1 mRNAs

  • Conduct proteomic analysis to measure protein abundance changes

• Stress recovery:

  • Investigate dynamics of eRF1iso2 during stress recovery phases

  • Compare with other translation factors like eRF3 and ABCE1

  • Treatment with translation inhibitors like puromycin or emetine can provide insights into the dynamics of stress granule formation and disassembly

What is the role of eRF1 isoform 2 in nonsense-mediated decay (NMD) and readthrough regulation?

To investigate eRF1iso2's role in NMD and readthrough regulation:

• NMD reporter assays:

  • Use reporters containing premature termination codons (PTCs)

  • Measure NMD efficiency upon eRF1iso2 overexpression or depletion

  • Research indicates eRF1 is a component of the SURF complex involved in NMD of mRNAs containing premature stop codons

• Readthrough quantification:

  • Employ dual luciferase reporters separated by stop codons in various contexts

  • Measure readthrough frequency with altered eRF1iso2 levels

  • Studies show eRF1 autoregulation can involve readthrough mechanisms

• UPF1 interaction studies:

  • Investigate eRF1iso2's ability to recruit UPF1 to ribosomes stalled at premature stop codons

  • Compare with eRF1iso1's activity in similar contexts

  • The SURF complex (which includes eRF1) recruits UPF1 to stalled ribosomes in NMD

• Pharmaceutical modulation:

  • Test compounds like SRI-41315 that affect eRF1 levels and readthrough

  • Research has shown that reducing eRF1 abundance can promote readthrough and may serve as a therapeutic approach for premature termination codon suppression

How can I investigate the differential tissue expression and regulation of eRF1 isoform 2?

To study tissue-specific expression and regulation of eRF1iso2:

• Tissue expression profiling:

  • Analyze publicly available RNA-seq datasets for tissue-specific expression of eRF1 transcript variants

  • Perform Western blot analysis on tissue panels using antibodies that can distinguish isoforms

  • Research indicates eRF1iso2 is expressed in human, rabbit, and mouse tissues

• Ribosome profiling analysis:

  • Examine ribosome profiling data to assess translation of different eRF1 isoforms

  • Studies show ribosome profiling of human embryonic stem cells can provide insights into isoform expression

• Splice variant analysis:

  • Design primers to specifically amplify different splice variants of eRF1

  • Quantify relative abundance in different tissues and developmental stages

  • Human eRF1 isoform 2 is encoded by multiple transcript variants (transcripts 2, 4, 5, and 6)

• Regulatory mechanism investigation:

  • Study the promoters of different eRF1 transcript variants

  • Investigate role of RNA-binding proteins in regulating splicing

  • Research indicates that plant eRF1 expression is controlled by a complex negative autoregulatory circuit involving features of the 3'UTR

How might eRF1 isoform 2 contribute to disease pathophysiology or therapeutic resistance?

Recent insights suggest several potential roles for eRF1iso2 in disease contexts:

• Cancer biology:

  • Analyze correlation between eRF1iso2 expression and cancer progression

  • Investigate whether altered stop codon recognition contributes to oncogenic mechanisms

  • Explore potential contributions to translation of truncated oncoproteins

• Genetic diseases with premature stop codons:

  • Examine whether eRF1iso2/eRF1iso1 ratios affect readthrough of disease-causing premature termination codons

  • Research indicates approaches that reduce eRF1 abundance may serve as potential therapeutic strategies for PTC suppression

  • Investigate synergy between eRF1iso2 modulation and readthrough-promoting drugs

• Viral infections:

  • Study the role of eRF1iso2 in viral translation termination and frameshifting

  • Investigate potential viral mechanisms that alter eRF1 splicing or stability

  • Research shows eRF1 is required for SHFL-mediated translation termination which inhibits programmed ribosomal frameshifting (-1PRF) of mRNA from viruses and cellular genes

• Therapeutic targeting:

  • Explore small molecules that could specifically modulate eRF1iso2 function

  • Investigate potential of isoform-specific targeting to affect termination efficiency

  • Studies show compounds like SRI-41315 can promote readthrough by diminishing eRF1 protein abundance through proteasomal degradation

What advanced techniques can be used to visualize eRF1 isoform 2 dynamics during translation in living cells?

Cutting-edge approaches to visualize eRF1iso2 dynamics include:

• Live-cell protein labeling:

  • Create fluorescent protein fusions or HaloTag/SNAP-tag fusions of eRF1iso2

  • Use super-resolution microscopy to track intracellular dynamics

  • Employ photoactivatable or photoconvertible tags for pulse-chase imaging

• Single-molecule imaging:

  • Apply techniques like single-molecule tracking or fluorescence correlation spectroscopy

  • Measure diffusion coefficients and binding kinetics in living cells

  • Compare dynamics of eRF1iso1 versus eRF1iso2 during normal translation and stress

• FRET-based interaction studies:

  • Design FRET pairs between eRF1iso2 and potential interaction partners

  • Monitor real-time association and dissociation events

  • Investigate conformational changes upon binding to ribosomes or eRF3

• Proximity labeling in living cells:

  • Use techniques like APEX2 or TurboID fused to eRF1iso2

  • Trigger biotinylation at defined timepoints to capture dynamic interactomes

  • Compare interactomes under different cellular conditions

These approaches can reveal the dynamic association of eRF1iso2 with ribosomes and its behavior during translation termination events.

Methodological Table: Comparative Analysis of eRF1 Isoform 2 Detection Methods

Key Research Models and Resources for eRF1 Isoform 2 Studies

• Cell lines with validated eRF1iso2 expression:

  • HeLa cells (human cervical cancer)

  • Human embryonic stem (hES) cells

  • Rabbit reticulocyte lysate (RRL)

  • Mouse Krebs-2 ascites cells

• Vectors and expression systems:

  • Bacterial expression systems for recombinant eRF1iso2 production

  • Mammalian expression vectors with tetracycline-inducible SBP-tagged eRF1

  • Dual luciferase reporters for readthrough quantification

• Validated antibodies:

  • Rabbit polyclonal antibodies against C-terminal domain of eRF1

  • Commercial antibodies suitable for Western blot (1:1000) and ICC/IF applications

• Specialized methodologies:

  • Reconstituted mammalian in vitro translation systems for functional studies

  • Selective Monosome-Seq for enrichment of eRF1-bound ribosomes

  • UPF1 dominant-negative (U1DN) agroinfiltration assay for NMD studies