ERF116 Antibody

What is ERF116 and why are antibodies against it important for plant research?

ERF116 (Uniprot: Q8GW17) is an Ethylene-responsive transcription factor belonging to the AP2/ERF transcription factor family in Arabidopsis thaliana. This protein likely functions as a transcriptional activator that binds to the GCC-box pathogenesis-related promoter element. ERF116 antibodies are critical research tools because:

• They enable detection, quantification, and characterization of ERF116 protein in plant tissues

• They facilitate the study of ERF116's role in plant stress responses and hormone signaling pathways

• They allow researchers to investigate ERF116's interactions with other proteins in transcriptional regulatory networks

• They help elucidate the functional role of ERF116 in plant development and physiology
  AP2/ERF family transcription factors like ERF116 have emerged as key regulators of several abiotic stresses and respond to multiple hormones , making antibodies against them essential for understanding plant stress response mechanisms.

What are the optimal applications for ERF116 antibody in plant molecular biology?

ERF116 antibody can be utilized in multiple experimental applications:

• Western Blotting: The primary application for detecting ERF116 protein expression levels. Recommended dilution is typically 1:1000 for optimal signal-to-noise ratio.

• Immunoprecipitation: To isolate ERF116 and its interacting protein partners to study protein-protein interactions and transcriptional complexes.

• Chromatin Immunoprecipitation (ChIP): To identify DNA-binding sites of ERF116 and investigate its role in transcriptional regulation during stress responses.

• Immunohistochemistry/Immunofluorescence: To visualize the subcellular localization and tissue-specific expression of ERF116, typically in the nucleus where it functions as a transcription factor.

• ELISA: For quantitative measurement of ERF116 protein levels in plant extracts.
  The choice of application should be guided by the specific research question and the validation data provided with the antibody.

How should researchers validate the specificity of ERF116 antibody?

Rigorous validation is essential for reliable research results. For ERF116 antibody, validation should include:

• Positive and negative controls:

  • Positive: Wild-type Arabidopsis thaliana tissue expressing ERF116

  • Negative: erf116 knockout/knockdown mutant plants

• Western blot analysis: Confirm a single band at the expected molecular weight (~50 kDa) with no non-specific binding.

• Pre-adsorption test: Pre-incubate the antibody with the immunizing peptide before use in the intended application. Signal elimination confirms specificity.

• Cross-reactivity assessment: Test against related ERF proteins (especially ERF107, ERF114, and ERF117 ) to determine specificity within the AP2/ERF family.

• Reproducibility testing: Perform technical and biological replicates to ensure consistent results across different experimental conditions.
  Remember that antibody validation should be performed for each specific application and experimental condition.

What are the critical factors for optimizing Western blot protocols with ERF116 antibody?

For optimal Western blot results with ERF116 antibody:

• Sample preparation:

  • Extract nuclear proteins where ERF116 is predominantly localized

  • Use phosphatase inhibitors as ERF transcription factors may be regulated by phosphorylation

  • Include protease inhibitors to prevent degradation

• Electrophoresis conditions:

  • Use 10-12% SDS-PAGE gels for optimal resolution around 50 kDa

  • Load 20-50 μg of total protein or 5-10 μg of nuclear extract

• Transfer parameters:

  • Semi-dry or wet transfer at 100V for 1 hour or 30V overnight

  • Use PVDF membrane (preferred over nitrocellulose for transcription factors)

• Blocking and antibody incubation:

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

  • Incubate with primary antibody (1:1000 dilution) overnight at 4°C

  • Use secondary antibody at 1:5000-1:10000 dilution for 1 hour at room temperature

• Detection optimization:

  • Enhanced chemiluminescence (ECL) detection is usually sufficient

  • Consider enhanced sensitivity ECL for low abundance samples
    Troubleshooting: If background is high, increase washing steps or reduce antibody concentration. If signal is weak, increase antibody concentration or protein loading.

How does the ERF116 antibody compare with antibodies for other AP2/ERF family members?

The AP2/ERF family contains 122 members in Arabidopsis thaliana and 139 in rice , making specificity a critical consideration:

• Use careful antibody selection based on epitope regions to minimize cross-reactivity

• Consider using epitope-tagged versions of the proteins for unambiguous detection

• Validate specificity through knockout/knockdown controls for each protein

• Use reciprocal immunoprecipitation to confirm protein interactions
  AP2/ERF proteins show conserved DNA binding domains but divergent activation/repression domains , which can be targeted for specific antibody generation.

What are the best methods for studying ERF116 post-translational modifications using antibodies?

ERF transcription factors are subject to various post-translational modifications that regulate their activity and stability:

• Phosphorylation detection:

  • Use phospho-specific antibodies if available

  • Alternatively, perform lambda phosphatase treatment before Western blotting to identify mobility shifts

  • AP2/ERF proteins can be phosphorylated by MAP kinases, SnRKs, or CK1

• Ubiquitination analysis:

  • Immunoprecipitate ERF116 under denaturing conditions

  • Probe with anti-ubiquitin antibodies

  • Use proteasome inhibitors (MG132) to stabilize ubiquitinated forms

• SUMOylation detection:

  • Immunoprecipitate with ERF116 antibody

  • Probe with anti-SUMO antibodies

  • Verify with SUMO protease treatment

• Acetylation assessment:

  • Immunoprecipitate ERF116

  • Probe with anti-acetyl-lysine antibodies

  • Confirm with HDAC inhibitor treatments
    The experimental approach should include careful sample preparation with appropriate inhibitors of modifying and demodifying enzymes to preserve the modifications of interest.

How can ERF116 antibody be used to study protein-protein interactions in transcriptional complexes?

ERF116 likely functions within transcriptional complexes. To study these interactions:

• Co-immunoprecipitation (Co-IP):

  • Immunoprecipitate ERF116 from plant nuclear extracts

  • Identify co-precipitating proteins by Western blot or mass spectrometry

  • Confirm interactions with reciprocal Co-IP

• Chromatin Immunoprecipitation followed by Mass Spectrometry (ChIP-MS):

  • Use ERF116 antibody to pull down chromatin-associated complexes

  • Identify DNA-bound protein partners by mass spectrometry

  • ERF-family proteins often interact with TPL/TPR co-repressors or other transcription factors

• Proximity-dependent biotin identification (BioID):

  • Create fusion proteins of ERF116 with a biotin ligase

  • Identify proximal proteins that become biotinylated

  • Use streptavidin pulldown followed by Western blot with ERF116 antibody

• Bimolecular Fluorescence Complementation (BiFC) validation:

  • Confirm interactions identified by antibody-based methods

  • Use ERF116 antibody to verify expression levels of fusion proteins
    The AP2/ERF family transcription factors can form complex regulatory networks, with both activation and repression functions dependent on protein interactions , making antibody-based interaction studies particularly valuable.

What are the key considerations when using ERF116 antibody for ChIP experiments?

Chromatin Immunoprecipitation (ChIP) with ERF116 antibody requires specific optimization:

• Crosslinking optimization:

  • Test different formaldehyde concentrations (0.75-1.5%)

  • Optimize crosslinking time (10-20 minutes)

  • Consider dual crosslinking with DSG followed by formaldehyde for improved transcription factor ChIP

• Chromatin fragmentation:

  • Sonicate to achieve fragments of 200-500 bp

  • Verify fragmentation by agarose gel electrophoresis

  • Over-sonication can damage epitopes

• Antibody validation for ChIP:

  • Perform ChIP-qPCR on known targets before ChIP-seq

  • Include IgG control and input samples

  • Test antibody amount (typically 2-5 μg per reaction)

• Data analysis considerations:

  • ERF proteins bind to GCC-box elements (AGCCGCC)

  • Compare ChIP-seq peaks with known ERF binding motifs

  • Integrate with RNA-seq data to correlate binding with gene expression

• Known targets for validation:

  • Genes involved in ethylene response

  • Stress-responsive genes

  • Pathogenesis-related genes
    The ERF family members can regulate different target genes despite similar DNA-binding domains, making antibody specificity crucial for accurate ChIP results.

How can researchers effectively use ERF116 antibody to study its role in plant stress responses?

AP2/ERF transcription factors are key regulators in abiotic stress responses . To study ERF116's specific role:

• Expression analysis under stress conditions:

  • Use Western blotting to quantify ERF116 protein levels under various stresses (drought, salt, cold, heat)

  • Compare protein expression with transcript levels (RT-qPCR)

  • Include time-course experiments to capture dynamic responses

• Subcellular localization changes:

  • Use immunofluorescence with ERF116 antibody to track potential nuclear-cytoplasmic shuttling during stress

  • Compare control and stress conditions

  • Co-stain with markers for nuclear compartments

• Post-translational modification profiling:

  • AP2/ERF proteins often undergo stress-induced phosphorylation

  • Use phospho-specific antibodies if available or analyze mobility shifts

  • Compare modifications across stress treatments

• Target gene regulation:

  • Perform ChIP-seq under control and stress conditions

  • Identify condition-specific binding sites

  • Correlate with transcriptome changes

• Protein complex remodeling:

  • Use co-immunoprecipitation to identify stress-specific interaction partners

  • Compare protein complexes under normal and stress conditions
    The dynamic nature of plant stress responses requires careful experimental design with appropriate time points and controls when using antibody-based detection methods.

What methodological approaches can resolve contradictory results when using ERF116 antibody?

Researchers may encounter contradictory results when using ERF116 antibody. Systematic troubleshooting includes:

• Antibody validation reassessment:

  • Repeat specificity tests using knockout/knockdown controls

  • Test multiple antibody lots

  • Consider epitope accessibility issues in different applications

• Sample preparation variables:

  • Standardize protein extraction protocols

  • Control for plant growth conditions and developmental stages

  • Consider tissue-specific expression patterns

• Technical controls:

  • Include loading controls for normalization

  • Use spike-in controls for quantitative applications

  • Implement biological and technical replicates

• Cross-validation with orthogonal methods:

  • Compare antibody-based results with tagged protein versions

  • Verify with mass spectrometry

  • Corroborate with genetic approaches (mutants, overexpression)

• Specific experimental variables:

  • For nuclear proteins like ERF116, ensure proper nuclear extraction

  • Consider dynamic post-translational modifications

  • Account for protein degradation during sample processing
    When contradictory results persist, consider that ERF116 may have context-dependent functions influenced by tissue type, developmental stage, or environmental conditions that affect antibody performance.

How can ERF116 antibody be used in comparative studies across plant species?

The AP2/ERF family is conserved across plant species, enabling comparative studies:

• Cross-reactivity analysis:

  • Test ERF116 antibody against homologs in related species

  • Consider the conservation of the epitope region across species

  • Perform Western blot analysis with samples from multiple species

• Experimental design for cross-species studies:

  • Include positive controls from Arabidopsis thaliana

  • Adjust protein loading for potential expression differences

  • Consider evolutionary distance when interpreting cross-reactivity

• Sequence alignment considerations:

  • The AP2/ERF domain is typically conserved (especially residues Gly-4, Arg-6, Glu-16, Trp-28, Leu-29, Gly-30, and Ala-38)

  • C-terminal regions may be more variable

  • Epitope mapping helps predict cross-reactivity

• Functional conservation testing:

  • Use ChIP with ERF116 antibody to compare binding sites across species

  • Correlate with expression data to assess functional conservation

  • Consider synteny of potential target genes
    The ERF family has expanded differently across plant lineages (122 in Arabidopsis, 139 in rice) , so careful interpretation of comparative results is essential.

What advanced techniques can enhance the sensitivity and specificity of ERF116 antibody-based detection?

For challenging experiments requiring enhanced detection:

• Signal amplification methods:

  • Tyramide signal amplification (TSA) for immunohistochemistry

  • Poly-HRP systems for Western blotting

  • Proximity ligation assay (PLA) for detecting protein interactions with high specificity

• Advanced microscopy techniques:

  • Super-resolution microscopy for detailed subcellular localization

  • Single-molecule tracking to study dynamics

  • FRET-based approaches to verify proximity of interaction partners

• Mass spectrometry integration:

  • Immunoprecipitation followed by MS/MS (IP-MS)

  • Selected reaction monitoring (SRM) for targeted quantification

  • Parallel reaction monitoring (PRM) for improved selectivity

• Microfluidic approaches:

  • Single-cell Western blotting

  • Microfluidic immunoprecipitation for limited samples

  • Automated multiplexed assays

• Artificial intelligence augmentation:

  • Machine learning algorithms for image analysis and antibody specificity prediction

  • Deep learning for pattern recognition in complex datasets

  • Computational modeling of antibody-antigen interactions to predict cross-reactivity These advanced techniques can be particularly valuable when studying low-abundance transcription factors like ERF116 or when analyzing small sample sizes from specific cell types.