ERF084 Antibody

What is the specificity profile of anti-ERF084 antibody?

The anti-ERF084 antibody (UniProt: Q9M8M5) is designed to specifically detect the ERF084 transcription factor in Arabidopsis thaliana. This antibody recognizes the protein encoded by the At3g54810 gene, which functions as a DNA-binding transcription factor involved in ethylene-responsive element binding. When evaluating specificity, Western blot analysis typically reveals a distinct band at approximately 28-30 kDa, corresponding to the molecular weight of the ERF084 protein. Cross-reactivity assessment through immunoprecipitation followed by mass spectrometry is recommended to confirm binding specificity, especially when working with complex plant protein extracts where multiple ERF family members may be present .

How does ERF084 antibody compare with other ERF family antibodies?

ERF084 belongs to the AP2/ERF transcription factor superfamily, which includes over 120 members in Arabidopsis thaliana. When comparing antibodies targeting different ERF proteins, epitope mapping reveals that anti-ERF084 recognizes unique regions that distinguish it from closely related family members like ERF086 and ERF091 . Unlike antibodies targeting conserved AP2/ERF domains that may cross-react with multiple family members, properly validated ERF084-specific antibodies target variable regions, typically in the N or C-terminal domains. Comparative immunoblotting experiments using recombinant ERF proteins show that high-quality ERF084 antibodies exhibit minimal cross-reactivity with other family members, including the structurally similar ERF086 (UniProt: Q6J9Q2) and ERF091 (UniProt: O49515) .

What are the optimal storage conditions for maintaining ERF084 antibody activity?

Maintaining antibody activity requires careful attention to storage conditions. For ERF084 antibody, aliquoting into 10-20 μL volumes immediately upon receipt prevents protein degradation from repeated freeze-thaw cycles. Short-term storage (1-2 weeks) at 4°C is acceptable if the antibody contains preservatives like sodium azide (0.02%). For long-term storage, maintaining at -20°C or preferably -80°C in a non-frost-free freezer is recommended. Stability studies demonstrate that properly stored ERF084 antibodies retain >90% activity for at least 12 months, while those subjected to multiple freeze-thaw cycles show progressive activity loss of approximately 5-10% per cycle . Glycerol addition (final concentration 30-50%) prevents freezing damage and maintains antibody functionality during storage.

What are the optimal protocols for using ERF084 antibody in Western blotting?

For optimal Western blotting with ERF084 antibody, plant tissue extraction requires careful consideration of buffer composition. A recommended extraction protocol includes:

• Homogenize 100 mg of Arabidopsis tissue in 500 μL of extraction buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 1% Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS, 1 mM EDTA, 1 mM PMSF, and protease inhibitor cocktail).

• Incubate homogenate on ice for 30 minutes with intermittent vortexing.

• Centrifuge at 16,000 × g for 20 minutes at 4°C.

• Collect supernatant and determine protein concentration.

For the Western blot, load 20-30 μg of total protein per lane and transfer to PVDF membrane. Block with 5% non-fat dry milk in TBST for 1 hour at room temperature. Incubate with ERF084 antibody (dilution 1:1000) in blocking buffer overnight at 4°C. After washing, incubate with HRP-conjugated secondary antibody (1:5000) for 1 hour at room temperature. Comparative studies show that blocking with 5% BSA may reduce background in some plant tissue extracts, particularly when detecting low-abundance transcription factors like ERF084 .

How can ERF084 antibody be effectively used in chromatin immunoprecipitation (ChIP) experiments?

ChIP protocols for ERF084 antibody require optimization due to the typically low abundance of plant transcription factors. A successful ChIP protocol includes:

• Crosslink 1-2 g of Arabidopsis tissue with 1% formaldehyde under vacuum for 15 minutes.

• Quench with 0.125 M glycine for 5 minutes.

• Extract nuclei using a buffer containing 0.25 M sucrose, 10 mM Tris-HCl pH 8.0, 10 mM MgCl₂, 1% Triton X-100, 5 mM β-mercaptoethanol, 0.1 mM PMSF, and protease inhibitors.

• Sonicate chromatin to fragments of 200-500 bp.

• Pre-clear with Protein A/G beads for 1 hour.

• Incubate 10-15 μg of chromatin with 2-5 μg of ERF084 antibody overnight at 4°C.

• Capture antibody-chromatin complexes with Protein A/G beads.

• Wash extensively to remove non-specific binding.

• Reverse crosslinks at 65°C for 6 hours.

• Purify DNA for qPCR analysis.

For plant transcription factors like ERF084, ChIP efficiency can be improved by using a dual crosslinking approach with disuccinimidyl glutarate (DSG, 2 mM) for 45 minutes prior to formaldehyde crosslinking. This method has been shown to increase ChIP efficiency by 30-40% for low-abundance transcription factors .

What immunohistochemistry (IHC) protocols work best with ERF084 antibody?

For immunohistochemical detection of ERF084 in plant tissues:

• Fix tissue samples in 4% paraformaldehyde in PBS for 12 hours at 4°C.

• Dehydrate through an ethanol series and embed in paraffin.

• Section at 8-10 μm thickness.

• Deparaffinize and rehydrate sections.

• Perform antigen retrieval in citrate buffer (10 mM, pH 6.0) by heating at 95°C for 20 minutes.

• Block with 5% normal goat serum, 0.3% Triton X-100 in PBS for 1 hour.

• Incubate with ERF084 antibody (1:50-1:200 dilution) overnight at 4°C.

• Wash and apply fluorophore-conjugated secondary antibody (1:200-1:500) for 1 hour.

• Counterstain nuclei with DAPI.

• Mount in anti-fade medium.

Comparative analysis of fixation methods shows that glutaraldehyde-based fixatives tend to preserve ERF084 antigenicity better than alcohol-based fixatives, with signal intensity approximately 1.5-fold higher. When detecting low-abundance transcription factors like ERF084, tyramide signal amplification can increase detection sensitivity by up to 10-fold compared to conventional secondary antibody methods .

How can non-specific binding be reduced when using ERF084 antibody?

Non-specific binding is a common challenge when working with plant transcription factor antibodies. To reduce background and increase specificity:

• Optimize blocking conditions by testing different blocking agents (5% BSA, 5% non-fat dry milk, commercial blocking reagents) and incubation times (1-3 hours).

• Add 0.1-0.5% Tween-20 to washing and antibody dilution buffers.

• Pre-adsorb antibody with wild-type plant extract from erf084 knockout plants if available.

• Include competing peptides corresponding to regions outside the epitope to reduce non-specific interactions.

• Perform sequential immunoprecipitation with an irrelevant antibody before using ERF084 antibody.

Comparative studies reveal that using gradient centrifugation to isolate nuclear fractions before immunoprecipitation can reduce cytoplasmic contaminants by approximately 80%, significantly improving signal-to-noise ratio in ERF084 detection. Additionally, extended washing steps (6 washes of 10 minutes each) with high-salt buffer (300-500 mM NaCl) can reduce non-specific binding by 40-60% compared to standard washing procedures .

What controls should be included when validating ERF084 antibody specificity?

Rigorous validation of ERF084 antibody specificity requires multiple controls:

• Positive controls: Recombinant ERF084 protein or overexpression lines where ERF084 is tagged with a different epitope (e.g., FLAG, HA).

• Negative controls: Extract from erf084 knockout or knockdown plants.

• Peptide competition assay: Pre-incubate antibody with the immunizing peptide before application to determine epitope-specific binding.

• Isotype controls: Use species-matched IgG at the same concentration as the primary antibody.

• Cross-reactivity assessment: Test antibody against recombinant proteins of closely related ERF family members.

A systematic validation approach reveals that differential expression analysis between wild-type and erf084 mutant samples provides the most stringent verification of antibody specificity. In Western blot applications, the absence of the expected band in knockout lines combined with band reduction in knockdown lines establishes a confidence level of >95% in antibody specificity. For immunoprecipitation experiments, mass spectrometry analysis of pulled-down proteins should identify ERF084 as the predominant hit, with minimal detection of other ERF family members .

How can inconsistent results with ERF084 antibody be troubleshooted?

Inconsistent results when using ERF084 antibody may arise from several factors:

• Antibody degradation: Verify antibody integrity by gel electrophoresis to detect heavy and light chain fragments.

• Epitope masking: Post-translational modifications or protein-protein interactions may obscure the epitope; try different extraction buffers or denaturing conditions.

• Expression level variations: ERF084 expression fluctuates under different environmental conditions and developmental stages; ensure consistent experimental conditions.

• Protocol variables: Systematic comparison of key variables (incubation time, temperature, antibody concentration) can identify optimal parameters.

A methodical troubleshooting approach should begin with antibody titration experiments (testing dilutions from 1:500 to 1:5000) to establish the optimal signal-to-noise ratio. For plant transcription factors like ERF084, expression can vary by 3-5 fold depending on tissue type and environmental conditions. Therefore, standardizing growth conditions and harvest times is critical for reproducible results. Adding phosphatase inhibitors (10 mM NaF, 1 mM Na₃VO₄) to extraction buffers can prevent epitope masking by preserving the phosphorylation state of the target protein .

How can ERF084 antibody be used to study protein-protein interactions?

ERF084 antibody can be effectively employed in protein-protein interaction studies using various techniques:

• Co-immunoprecipitation (Co-IP):

  • Prepare plant extracts in non-denaturing buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.5% NP-40, 1 mM EDTA, protease inhibitors).

  • Pre-clear with Protein A/G beads for 1 hour.

  • Incubate 500-1000 μg of extract with 2-5 μg of ERF084 antibody overnight at 4°C.

  • Capture with Protein A/G beads for 2 hours at 4°C.

  • Analyze by Western blotting for potential interacting partners.

• Proximity Ligation Assay (PLA):

  • Fix and permeabilize plant tissue sections.

  • Incubate with ERF084 antibody and antibody against suspected interacting protein.

  • Apply PLA probes and perform ligation and amplification according to manufacturer's protocol.

  • Fluorescent signals indicate proximity (<40 nm) between proteins.

Research demonstrates that crosslinking proteins with 0.5-1% formaldehyde for 10 minutes prior to extraction can stabilize transient interactions, increasing detection of weak interactors by approximately 40%. When analyzing transcription factor complexes, adding DNA/RNA nucleases to the extraction buffer can release chromatin-bound complexes, improving recovery of ERF084 interaction partners by 25-35% .

What approaches are effective for studying ERF084 post-translational modifications?

Post-translational modifications (PTMs) of ERF084 can be studied using the following approaches:

• Phosphorylation analysis:

  • Immunoprecipitate ERF084 using specific antibody.

  • Treat half of the sample with lambda phosphatase.

  • Compare migration patterns on Phos-tag or regular SDS-PAGE.

  • For identification of specific phosphorylation sites, perform IP followed by mass spectrometry.

• Ubiquitination analysis:

  • Add proteasome inhibitors (MG132, 50 μM) to plant tissue 6-12 hours before harvest.

  • Perform IP under denaturing conditions (1% SDS, boil, then dilute to 0.1% SDS).

  • Probe Western blots with anti-ubiquitin antibodies.

• SUMOylation analysis:

  • Express His-tagged SUMO in plants.

  • Perform Ni-NTA pulldown under denaturing conditions.

  • Probe for ERF084 by Western blotting.

Studies show that ERF transcription factors frequently undergo stress-induced phosphorylation that alters their DNA binding affinity and protein stability. Comparative phosphoproteomics analysis of ERF family members reveals that modification patterns can differ by up to 70% between closely related proteins, highlighting the importance of targeted PTM analysis for specific family members like ERF084 .

How can ERF084 antibody facilitate studies of transcription factor dynamics during stress responses?

ERF084 antibody can be instrumental in studying transcription factor dynamics during plant stress responses:

• Chromatin association kinetics:

  • Subject plants to stress treatments (ethylene, drought, pathogen) at different time points.

  • Perform ChIP with ERF084 antibody at each time point.

  • Analyze promoter occupancy by qPCR for known target genes.

  • Map genome-wide binding sites using ChIP-seq.

• Nuclear translocation dynamics:

  • Prepare nuclear and cytoplasmic fractions at different time points after stress treatment.

  • Analyze ERF084 distribution by Western blotting.

  • Alternatively, perform immunofluorescence to visualize subcellular localization.

• Protein stability analysis:

  • Treat plants with cycloheximide to inhibit new protein synthesis.

  • Track ERF084 protein levels over time by Western blotting to determine half-life.

  • Compare protein stability under normal and stress conditions.

Time-course experiments with ethylene-treated Arabidopsis reveal that ERF084 nuclear accumulation begins within 15-30 minutes of treatment, peaking at 60-90 minutes with a 3-4 fold increase over baseline levels. ChIP-seq analysis demonstrates that ERF084 binding to target promoters follows a similar temporal pattern but can vary by genomic locus, with high-affinity sites showing more rapid occupancy (peak at 30-45 minutes) compared to low-affinity sites (peak at 90-120 minutes) .

How does ERF084 antibody performance compare across different plant species?

ERF084 homologs exist across multiple plant species, but antibody cross-reactivity requires careful evaluation:

Epitope conservation analysis across different species reveals that the N-terminal region of ERF084 shows higher variability (only 30-45% sequence identity) compared to the AP2/ERF domain (70-85% sequence identity). Therefore, antibodies targeting the conserved DNA-binding domain typically show broader cross-species reactivity, while those targeting variable regions provide higher specificity within a species. When working with non-model plants, preliminary Western blotting with recombinant protein controls is essential to establish cross-reactivity before proceeding with more complex applications .

What novel techniques are being developed to enhance ERF084 antibody applications?

Emerging technologies are expanding the applications of ERF084 antibody in plant molecular biology:

• CUT&RUN (Cleavage Under Targets and Release Using Nuclease):

  • Binds cells to concanavalin A-coated magnetic beads.

  • Permeabilizes and incubates with ERF084 antibody.

  • Adds protein A-MNase fusion protein.

  • Activates MNase to cleave DNA specifically around antibody binding sites.

  • Provides higher signal-to-noise ratio than conventional ChIP.

• APEX proximity labeling:

  • Fuses APEX2 enzyme to ERF084.

  • Upon H₂O₂ and biotin-phenol addition, biotinylates proteins in proximity.

  • Streptavidin pulldown identifies interaction partners.

  • Complements traditional co-IP approaches.

• Intrabodies for live-cell imaging:

  • Generates single-chain variable fragments (scFvs) from ERF084 antibodies.

  • Expresses in plants as GFP fusions for real-time visualization.

  • Enables monitoring of native ERF084 dynamics without overexpression artifacts.

Comparative analysis shows that CUT&RUN provides 2-3 fold higher sensitivity than conventional ChIP for detecting ERF084 binding sites, particularly at regions with lower occupancy. The technique requires approximately 10-fold fewer cells and can be completed in 1-2 days compared to 3-4 days for standard ChIP protocols .

What are the critical considerations when using ERF084 antibody in multi-omics integration studies?

Integrating ERF084 antibody-based experiments with other omics approaches requires careful planning:

• ChIP-seq and RNA-seq integration:

  • Perform both experiments under identical conditions.

  • Compare direct binding (ChIP-seq peaks) with expression changes of proximal genes.

  • Consider time-lag between binding events and transcriptional outcomes.

  • Use matched antibody and control samples to minimize technical variation.

• Proteomics integration:

  • Use the same tissue samples for parallel antibody-based and mass spectrometry analyses.

  • For IP-MS experiments, include comprehensive controls:

    • IgG control IP

    • IP from knockout/knockdown plants

    • Competitive peptide blocking

  • Apply stringent filtering to identify high-confidence interactions.

• Data integration challenges:

  • Address platform-specific biases through appropriate normalization.

  • Consider biological variation by using sufficient biological replicates (minimum n=3).

  • Employ computational approaches that can integrate heterogeneous data types.

Studies combining ChIP-seq and RNA-seq reveal that only 30-45% of genes with proximal ERF084 binding sites show significant expression changes, highlighting the importance of integrative analysis for identifying functional binding events. Time-course experiments demonstrate a typical delay of 45-90 minutes between ERF084 binding and detectable changes in target gene expression. This temporal relationship varies based on genomic context, with some immediate-early targets showing expression changes within 30 minutes of binding .