ERF003 Antibody

What is ERF003 and what experimental systems can ERF003 antibody be used in?

ERF003 antibodies are primarily used in plant research, with demonstrated reactivity in Arabidopsis systems . These antibodies have been validated for Western Blot (WB) and ELISA applications, making them suitable for detecting native ERF003 protein expression in plant extracts . Unlike translation termination factor antibodies (such as eRF3/GSPT1) that work across multiple mammalian species, ERF003 antibodies show specific reactivity with plant samples, requiring careful experimental design when working across species boundaries.

What are the recommended sample preparation protocols for Western blot analysis using ERF003 antibody?

For optimal Western blot results using ERF003 antibody, consider these methodological approaches:

• Extract plant tissue in buffer containing 50mM Tris-HCl (pH 7.5), 150mM NaCl, 1% Triton X-100, and protease inhibitors

• Clarify lysates by centrifugation (15,000×g, 15 minutes at 4°C)

• Determine protein concentration using standard Bradford or BCA assays

• Load 20-50μg total protein per lane on SDS-PAGE gels (10-12%)

• Use dilution ratios similar to other plant antibodies (1:1000-1:2000)

• Include positive controls from Arabidopsis extracts

This protocol draws on standard practices for plant antibodies, as specific ERF003 dilution recommendations must be optimized for each experimental system.

How should ERF003 antibody be stored and handled to maintain optimal activity?

To maintain ERF003 antibody functionality:

• Store at -20°C in a non-frost-free freezer

• Avoid repeated freeze-thaw cycles by preparing single-use aliquots

• Store in buffer containing glycerol (typically 50%) to prevent freeze damage

• Add preservatives like sodium azide (0.02%) for long-term storage

• Follow manufacturer's recommendations for stability duration

• Monitor performance over time using positive control samples

Similar antibody products are typically stable for at least 12 months when stored properly .

What controls should be included when validating ERF003 antibody specificity?

Proper validation requires multiple controls:

These validation strategies ensure experimental rigor and reproducibility when working with ERF003 antibodies in research contexts.

How can ERF003 antibody be optimized for immunoprecipitation experiments?

For successful immunoprecipitation (IP) of ERF003:

• Use mild lysis conditions to preserve protein-protein interactions (150-300mM NaCl, 0.5-1% NP-40 or Triton X-100)

• Pre-clear lysates with Protein A/G beads to reduce non-specific binding

• Optimize antibody-to-lysate ratio (typically starting with 2-5μg antibody per 500μg total protein)

• Consider crosslinking the antibody to beads to prevent antibody contamination in eluates

• Use gentle elution conditions (low pH glycine buffer or competitive elution with immunizing peptide)

• Validate IP efficiency by Western blot analysis of input, flow-through, and eluate fractions

This methodology draws on established approaches for plant protein immunoprecipitation, though specific optimization for ERF003 may be required based on protein abundance and antibody affinity.

What considerations should be made when detecting post-translational modifications of ERF003?

When investigating post-translational modifications:

• Use phosphatase inhibitors (sodium orthovanadate, sodium fluoride) in extraction buffers when studying phosphorylation

• Consider specialized extraction protocols for studying ubiquitination (include deubiquitinase inhibitors)

• Validate modification-specific signals using treatment controls (phosphatase treatment, deubiquitinase treatment)

• Use protease inhibitor cocktails optimized for plant tissue extraction

• Consider using modification-specific antibodies in conjunction with ERF003 antibody

• Employ mass spectrometry validation of detected modifications

These approaches help ensure accurate detection and characterization of ERF003 post-translational modifications in experimental systems.

How do the methodologies for ERF003 antibody applications compare to those for translation factor antibodies?

When comparing methodological approaches between plant ERF003 antibodies and well-characterized translation factor antibodies such as eRF3:

This comparison highlights the extensive characterization of mammalian translation factor antibodies compared to plant ERF003 antibodies, providing methodological insights that might be applicable across systems.

What are the key considerations when developing co-localization experiments using ERF003 antibody?

For successful co-localization studies:

• Select appropriate fixation methods (4% paraformaldehyde for 15-20 minutes works for many plant proteins)

• Optimize permeabilization conditions to maintain cellular architecture while allowing antibody access

• Use multi-fluorophore labeling with spectrally distinct secondary antibodies

• Include appropriate controls:

  • Single-antibody controls to assess bleed-through

  • Peptide competition controls to confirm specificity

  • Secondary-only controls to assess background

• Use confocal microscopy with sequential scanning to minimize crosstalk

• Quantify co-localization using established metrics (Pearson's coefficient, Manders' overlap coefficient)

These methodological considerations help ensure reliable co-localization results when studying ERF003 alongside other proteins of interest.

How does ERF003 antibody development methodology compare to bispecific antibody approaches?

The development strategies for research-grade monospecific antibodies like ERF003 differ significantly from bispecific therapeutic antibodies:

• Design approach:

  • Research antibodies like ERF003: Generated against single epitope regions using traditional immunization methods

  • Bispecific antibodies: Engineered using sophisticated approaches like DART (Dual-Affinity Re-Targeting) or BiTE (Bispecific T-cell Engager) platforms

• Production considerations:

  • Research antibodies: Typically produced through hybridoma technology or recombinant expression

  • Bispecific antibodies: Require specialized linker engineering to connect two distinct binding domains with optimized (G4S)n linkers

• Validation requirements:

  • Research antibodies: Validated for basic research applications (WB, ELISA, etc.)

  • Bispecific antibodies: Require extensive functional validation for therapeutic efficacy

While ERF003 antibodies follow traditional monospecific development approaches, understanding advanced antibody engineering can inform research applications requiring dual targeting capabilities.

What experimental design considerations should be made when studying protein-protein interactions using ERF003 antibody?

A robust experimental design for studying ERF003 interactions should include:

• Multiple complementary approaches:

  • Co-immunoprecipitation with ERF003 antibody

  • Reciprocal co-IP with antibodies against suspected interaction partners

  • Proximity ligation assay (PLA) for in situ detection of interactions

  • Fluorescence resonance energy transfer (FRET) for direct interaction measurement

• Appropriate controls:

  • IgG control immunoprecipitations

  • Input samples (5-10% of lysate used for IP)

  • Non-interacting protein controls

  • Competitive binding with excess antigen

• Stringency optimization:

  • Test multiple buffer conditions (varying salt and detergent concentrations)

  • Compare native versus crosslinked conditions

  • Consider size exclusion chromatography validation

How can researchers effectively troubleshoot non-specific binding issues with ERF003 antibody?

When encountering non-specific binding:

• Optimization strategies:

  • Increase blocking stringency (5% BSA or 5% milk, consider adding 0.1-0.5% Tween-20)

  • Test different blocking agents (BSA, milk, normal serum from secondary antibody host species)

  • Optimize antibody concentration through titration experiments

  • Increase washing stringency (higher salt concentration, longer/more frequent washes)

• Sample preparation modifications:

  • Pre-clear lysates with Protein A/G beads before immunoprecipitation

  • Use gradient gels to improve protein separation

  • Consider membrane pre-treatment methods

• Validation approaches:

  • Compare results from different antibody lots

  • Perform peptide competition assays

  • Include knockout/knockdown controls where possible

These methodological refinements can significantly improve signal-to-noise ratio when working with ERF003 antibody.

What considerations should be made when designing CRISPR/Cas9 validation systems for ERF003 antibody specificity?

CRISPR/Cas9-based validation requires:

• Guide RNA design:

  • Target early exons to ensure complete protein disruption

  • Design multiple guides to increase knockout efficiency

  • Check for off-target effects using prediction algorithms

  • Include targeting controls (non-targeting guides)

• Validation methodology:

  • Confirm genetic modification by sequencing

  • Validate knockout at mRNA level using RT-qPCR

  • Test antibody specificity in wild-type versus knockout samples

  • Include protein restoration experiments (rescue with cDNA expression)

• Controls and quantification:

  • Include wild-type controls from same genetic background

  • Quantify signal reduction in knockout lines

  • Assess non-specific bands that persist in knockout samples

This rigorous approach provides definitive evidence for antibody specificity against ERF003 target protein.

How can mass spectrometry complement antibody-based detection of ERF003?

Integrating mass spectrometry with antibody techniques:

• Sample preparation workflows:

  • Immunoprecipitate ERF003 using validated antibody

  • Perform in-gel or in-solution digestion

  • Consider enrichment strategies for post-translational modifications

• MS analysis approaches:

  • Use targeted Multiple Reaction Monitoring (MRM) for quantification

  • Employ data-dependent acquisition for discovery of modifications

  • Consider crosslinking mass spectrometry (XL-MS) for interaction studies

• Validation strategies:

  • Confirm antibody-detected modifications with MS data

  • Use synthetic peptide standards for absolute quantification

  • Correlate Western blot and MS-based quantification results

This complementary approach provides orthogonal validation of antibody-based findings while offering deeper insights into ERF003 biology.

What methodological approaches should be considered when studying ERF003 in different subcellular compartments?

For comprehensive subcellular localization studies:

• Fractionation approaches:

  • Differential centrifugation protocols optimized for plant cells

  • Density gradient separation for membrane compartments

  • Specific extraction protocols for nuclear, cytoplasmic, and membrane fractions

• Immunofluorescence strategies:

  • Co-staining with established organelle markers

  • Super-resolution microscopy for precise localization

  • Live-cell imaging with fluorescently-tagged ERF003 for dynamics studies

• Biochemical validation:

  • Western blot analysis of fractionated samples

  • Protease protection assays for membrane topology

  • Activity assays in isolated compartments

This multi-faceted approach provides comprehensive evidence for ERF003 localization and potential translocation under different cellular conditions.