ERF021 Antibody

What is ERF021 and what is its biological role in plants?

ERF021 belongs to the Ethylene Response Factor (ERF) family in plants, specifically in Arabidopsis thaliana. As a plant transcription factor, it's involved in stress response pathways and developmental processes. The protein contains an ETS domain and functions as part of the plant's transcriptional regulation system. Understanding its role provides insights into plant adaptation mechanisms to environmental stressors and development regulation .

What are the key specifications of commercially available ERF021 antibodies?

ERF021 antibodies are typically rabbit polyclonal antibodies raised against recombinant Arabidopsis thaliana ERF021 protein. They are supplied in liquid form containing PBS (pH 7.2) with 40-50% glycerol and 0.03% Proclin 300 as a preservative. Most are affinity-purified and designed for research applications including ELISA and Western Blotting. The typical product is purified using antigen affinity methods and is validated for Arabidopsis thaliana reactivity .

How does ERF021 antibody differ from other ERF family antibodies?

ERF021 antibody specifically targets the ERF021 protein in Arabidopsis thaliana, distinguishing it from other ERF family antibodies like ERF003, ERF025, or ERF036. Each antibody in this family is raised against different immunogens corresponding to specific amino acid sequences unique to each ERF protein. For example, commercially available ERF021 antibodies are developed against a recombinant protein corresponding to Arabidopsis thaliana ERF021, while other ERF antibodies target different epitopes specific to their respective proteins .

What are the validated applications for ERF021 antibody in plant research?

ERF021 antibody has been validated primarily for ELISA and Western Blotting applications in plant research. For Western Blotting, researchers typically use a dilution range similar to other plant ERF antibodies (typically 1:500-1:2000). The antibody is particularly useful for studying transcription factor expression and localization in Arabidopsis thaliana, making it valuable for research on plant stress responses and developmental biology .

What is the recommended protocol for Western Blotting using ERF021 antibody?

For optimal Western Blotting results with ERF021 antibody:

• Prepare protein samples by diluting in Laemmli buffer (with or without mercaptoethanol)

• Heat samples at 95°C for 5 minutes

• Separate proteins on 8-12% electrophoresis gels (run at 100V for approximately 2 hours)

• Transfer proteins to PVDF membrane

• Block membrane with PBS-T/1% BSA for 1 hour at 37°C

• Incubate with primary ERF021 antibody at recommended dilution (typically 1:500-1:2000) overnight at 4°C

• Wash with PBS-T (4 times)

• Incubate with secondary antibody (anti-rabbit HRP conjugate) at 1:5000-1:40000 dilution for 1 hour at 37°C

• Wash with PBS-T (4 times)

• Develop using chemiluminescence detection system

How can researchers optimize immunofluorescence experiments using ERF021 antibody?

For immunofluorescence optimization with ERF021 antibody:

• Fix samples in 4% paraformaldehyde at room temperature for 15-20 minutes

• Perform antigen retrieval if needed (particularly for paraffin-embedded sections)

• Block non-specific binding with appropriate blocking buffer (3-5% BSA in PBS)

• Dilute ERF021 antibody at 1:100-1:1000 in antibody dilution buffer

• Incubate at 4°C overnight or at room temperature for 1-2 hours

• Wash thoroughly with PBS (3-5 times, 5 minutes each)

• Apply appropriate fluorophore-conjugated secondary antibody

• For nuclear transcription factor visualization, co-stain with DAPI

• Include positive controls (Arabidopsis tissues with known ERF021 expression)

• Use confocal microscopy for optimal visualization of nuclear localization

How can researchers validate ERF021 antibody specificity in their experimental system?

Validating ERF021 antibody specificity requires multiple approaches:

• Immunoblotting with recombinant protein: Test antibody against purified recombinant ERF021 protein as a positive control

• Knockout/knockdown controls: Use ERF021 knockout or knockdown plant lines as negative controls

• Peptide competition assay: Pre-incubate antibody with excess immunizing peptide to confirm signal specificity

• Cross-reactivity assessment: Test against related ERF family proteins to confirm specificity

• Multiple antibody approach: Compare results with other antibodies targeting different epitopes of ERF021

• Mass spectrometry validation: Confirm identity of immunoprecipitated proteins by mass spectrometry

Researchers should document specificity through comparison with published expression patterns and molecular weight verification on Western blots (expected MW can be predicted from the amino acid sequence) .

What strategies should be employed when studying ERF transcription factor localization patterns?

When investigating ERF021 localization:

• Subcellular fractionation: Separate nuclear, cytoplasmic, and membrane fractions before immunoblotting

• Co-localization studies: Combine ERF021 antibody with markers for specific subcellular compartments

• Time-course experiments: Monitor localization changes following treatment with plant hormones (especially ethylene)

• Stress response dynamics: Track localization shifts under various abiotic stresses

• Live-cell imaging: Consider creating fluorescent protein fusions to complement antibody studies

• ChIP experiments: Use ERF021 antibody for chromatin immunoprecipitation to identify DNA binding sites

Successful localization studies require careful optimization of fixation protocols to preserve nuclear architecture while maintaining epitope accessibility .

How do post-translational modifications affect ERF021 antibody recognition?

Post-translational modifications (PTMs) can significantly impact antibody recognition of ERF021:

• Phosphorylation: ERF transcription factors are often regulated by phosphorylation, which may mask or reveal epitopes

• Epitope accessibility: Conformational changes due to PTMs may alter antibody binding efficiency

• Molecular weight shifts: Researchers should be aware that phosphorylated forms of ERF021 may show higher apparent molecular weights on Western blots

• Phosphatase treatment: Compare antibody recognition before and after phosphatase treatment to assess phosphorylation dependence

• PTM-specific antibodies: Consider using phospho-specific antibodies if studying activation states

• Nuclear translocation: Many ERFs show nuclear translocation following phosphorylation events

Understanding the relationship between PTMs and antibody recognition is critical for accurate interpretation of experimental results, particularly when studying signaling pathways .

What are the most common issues when using ERF021 antibody and how can they be resolved?

Proper experimental controls are essential for troubleshooting, including positive control samples with known ERF021 expression and negative controls using pre-immune serum or isotype controls .

What are the optimal storage and handling conditions for maintaining ERF021 antibody activity?

To maintain optimal ERF021 antibody activity:

• Store at 4°C for short-term use (up to 2 weeks)

• For long-term storage, keep at -20°C or -80°C

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

• Add carrier protein (BSA, 0.1-1%) if diluting for storage

• Include preservatives (0.02-0.03% sodium azide or Proclin 300) for diluted solutions

• Store in glycerol buffers (typically 40-50% glycerol) to prevent freezing damage

• Never store diluted antibody solutions for extended periods

• Monitor pH stability (optimal range: 7.0-7.4)

• Keep antibody vials upright and avoid surface contamination

• Document all freeze-thaw cycles and storage conditions for reproducibility

How should researchers compare data obtained using different lots of ERF021 antibody?

When comparing data across different antibody lots:

• Lot validation: Test each new lot against a reference sample with known ERF021 expression

• Signal quantification: Use densitometry to compare signal intensity between lots

• Standard curves: Generate standard curves using recombinant protein for each lot

• Internal loading controls: Normalize all data to consistent loading controls

• Archival samples: Maintain reference samples for long-term projects

• Epitope confirmation: Verify that different lots recognize the same epitope region

• Experimental replication: Perform key experiments with both old and new lots in parallel

• Documentation: Record lot numbers, dilutions, and performance metrics

Researchers should report antibody lot numbers in publications and be cautious when directly comparing quantitative data obtained with different lots .

How can phosphorylation status of ERF transcription factors be monitored using antibody-based approaches?

To monitor ERF021 phosphorylation:

• Phospho-specific antibodies: When available, these directly detect specific phosphorylated residues

• Mobility shift assays: Phosphorylated ERFs often show reduced electrophoretic mobility

• Phosphatase treatment: Compare antibody recognition before and after phosphatase treatment

• Phos-tag™ gels: Use specialized acrylamide gels that enhance separation of phosphorylated proteins

• 2D gel electrophoresis: Separate phospho-isoforms based on charge differences

• Immunoprecipitation followed by phospho-staining: IP with ERF021 antibody followed by Pro-Q Diamond staining

• Mass spectrometry analysis: For definitive identification of phosphorylation sites

Phosphorylation analysis is particularly relevant as ERF transcription factors are often regulated by MAPK cascades in response to stress conditions .

What comparative approaches can be used to study ERF021 in relation to other ERF family members?

For comparative ERF family studies:

• Multiple antibody immunoblotting: Probe identical samples with different ERF-specific antibodies

• Cross-reactivity testing: Determine if ERF021 antibody recognizes related family members

• Expression correlation analysis: Compare expression patterns across different conditions

• Co-immunoprecipitation: Investigate protein-protein interactions between ERF family members

• ChIP-seq comparison: Identify shared and unique DNA binding sites between ERF proteins

• Phylogenetic analysis: Map antibody epitope conservation across the ERF family

• Knockout/knockdown phenotyping: Compare phenotypes of different ERF mutants

This approach helps establish functional redundancy or specialization among ERF family members and places ERF021 in the broader context of plant transcriptional regulation .

How should researchers interpret contradictory results between antibody-based detection and transcript level measurements?

When antibody detection and transcript data conflict:

• Post-transcriptional regulation: Investigate miRNA targeting or transcript stability

• Translational control: Examine polysome association of ERF021 mRNA

• Protein stability: Assess proteasome-dependent degradation rates

• Epitope masking: Consider if protein interactions or modifications mask antibody epitopes

• Antibody specificity: Re-validate antibody specificity with appropriate controls

• Subcellular localization changes: Determine if protein redistribution affects extraction efficiency

• Temporal dynamics: Implement time-course studies to capture expression/translation delays

Such discrepancies often reveal important regulatory mechanisms and should be investigated thoroughly rather than dismissed as technical errors. Protein-transcript correlation analysis across multiple conditions can provide valuable insights into ERF021 regulation .

How can ERF021 antibody be applied in chromatin immunoprecipitation (ChIP) studies?

For successful ChIP experiments with ERF021 antibody:

• Crosslinking optimization: Test different formaldehyde concentrations (typically 1-3%) and incubation times

• Sonication parameters: Optimize sonication to generate 200-500 bp DNA fragments

• Antibody amount: Use 5-10 μg of ERF021 antibody per immunoprecipitation

• Chromatin amount: Start with approximately 10 μg of chromatin per IP

• Pre-clearing step: Include pre-clearing with protein A/G beads to reduce background

• Controls: Include no-antibody controls and preferably IgG controls

• Positive control regions: Target known ERF-binding promoters as positive controls

• Elution conditions: Optimize elution buffer composition and incubation times

• qPCR validation: Validate enrichment at candidate loci before proceeding to sequencing

The optimal antibody:chromatin ratio may require empirical determination for each experimental system. For whole-genome approaches, ChIP-seq library preparation should be optimized for the typically small amounts of immunoprecipitated DNA .

What methodological considerations are important when using ERF021 antibody in plant stress response studies?

For plant stress response studies:

• Timing is critical: Establish a detailed time-course to capture transient ERF021 expression/activation

• Tissue specificity: Different plant tissues may show distinct ERF021 responses

• Stress intensity standardization: Carefully control stress conditions for reproducibility

• Multiple stress types: Compare ERF021 response across different abiotic stressors

• Hormone interactions: Consider ethylene, jasmonate, and ABA crosstalk in experimental design

• Subcellular fractionation: Monitor potential translocation between cellular compartments

• Protein-protein interactions: Investigate stress-induced changes in interaction partners

• Post-translational modifications: Assess changes in phosphorylation status during stress response

Researchers should also consider developmental stage effects, as stress responses often vary based on plant age and developmental phase .

How can researchers integrate antibody-based detection with proteomics approaches for comprehensive ERF021 analysis?

For integrated antibody-proteomics approaches:

• Immunoprecipitation-mass spectrometry (IP-MS): Use ERF021 antibody to pull down protein complexes for MS analysis

• Proximity labeling: Combine with BioID or APEX2 approaches to identify neighboring proteins

• Crosslinking-IP (CLIP): Identify RNA interactions of ERF021 protein

• Selected reaction monitoring (SRM): Develop targeted MS assays for ERF021 quantification

• Parallel reaction monitoring (PRM): Enhance sensitivity for ERF021 detection in complex samples

• Post-translational modification mapping: Identify specific modified residues by MS after IP

• Comparison with total proteomics data: Contextualize ERF021 behavior within the global proteome

This integration allows researchers to move beyond simple detection toward understanding ERF021's place in complex regulatory networks and identifying novel interaction partners that may mediate its biological functions .