ERF120 Antibody

What is ERF120 and why is it important in plant research?

ERF120 (Q9SK67) is an ethylene response factor transcription factor in Arabidopsis thaliana that belongs to the ERF family. This family plays critical roles in plant immunity and stress responses. As described in research on related ERFs, these transcription factors are integrators of hormonal pathways and directly responsible for the transcriptional regulation of several jasmonate (JA)/ethylene (ET)-responsive defense genes . While specific data on ERF120 is limited, studies of other ERF family members (like ERF96) show they bind to promoter regions of JA/ET-dependent defense genes through GCC elements to regulate immune responses .

What are the standard applications for ERF120 antibody?

The ERF120 antibody (CSB-PA882889XA01DOA) is designed for several experimental applications:

• ELISA (Enzyme-Linked Immunosorbent Assay): For quantitative detection of ERF120 in plant samples

• Western Blot (WB): For identification and semi-quantitative analysis of ERF120 protein expression levels

The antibody is raised in rabbits against recombinant Arabidopsis thaliana ERF120 protein and is purified using antigen affinity methods . As with similar plant transcription factor antibodies, optimal dilution ratios should be determined experimentally for each application.

How should samples be prepared for optimal ERF120 detection?

For optimal detection of ERF120, samples should be prepared as follows:

• For protein extraction: Use HNTG buffer [50 mM Hepes (pH7.5), 150 mM NaCl, 10% glycerol, 1% Triton X-100, and 5 mM EGTA] or RIPA lysis buffer supplemented with protease and phosphatase inhibitors .

• For Western blot applications:

  • Denature samples at 95°C for 5 minutes in standard SDS sample buffer

  • Load 20-50 μg of total protein on SDS-PAGE gels

  • Transfer to suitable membranes (PVDF or nitrocellulose)

  • Block with 5% non-fat dry milk in TBST (similar to procedures used for other plant antibodies)

• For immunofluorescence:

  • Fix tissue samples with 4% paraformaldehyde

  • Permeabilize with 0.1% Triton X-100

  • Use appropriate secondary antibodies (typically Goat Anti-Rabbit IgG conjugated with fluorophores)

How can ERF120 antibody be used to study plant immunity pathways?

When designing experiments to investigate ERF120's role in plant immunity pathways, consider the following methodological approach:

• Pathogen challenge experiments:

  • Challenge Arabidopsis plants with pathogens (such as Botrytis cinerea or Pseudomonas syringae)

  • Collect tissue samples at various time points post-infection

  • Use ERF120 antibody to detect changes in protein expression via Western blot

  • Correlate ERF120 expression with defense gene activation (e.g., PDF1.2, PR3, PR4)

• Co-immunoprecipitation studies:

  • Use ERF120 antibody to pull down protein complexes

  • Analyze interacting partners through mass spectrometry

  • Validate interactions with known immunity regulators like ORA59 or EIN3

Research on related ERF transcription factors shows they can form positive feedback loops with other TFs to boost expression of defense genes . For example, ERF96 and ORA59 form a positive feedback loop to enhance JA/ET-dependent gene expression during necrotrophic pathogen infection.

What controls and validation steps are essential when using ERF120 antibody?

To ensure experimental validity when using ERF120 antibody, implement these critical controls:

• Antibody specificity validation:

  • Include a no-primary antibody control

  • Test on erf120 knockout/knockdown plant lines (expect reduced or absent signal)

  • Perform peptide competition assay using the immunogen peptide

  • Compare expression patterns with published transcriptomic data

• Experimental controls:

  • Include untreated samples as negative controls

  • Use samples with known ERF expression changes as positive controls

  • For phosphorylation studies, include alkaline phosphatase-treated samples

  • For hormone treatment studies, validate with established markers of hormone response

• Signal validation:

  • Confirm band size (predicted molecular weight)

  • Validate subcellular localization patterns align with known ERF distribution (typically nuclear)

How does ERF120 compare to other ERF family members in experimental applications?

When designing studies comparing multiple ERF family members, consider these methodological approaches:

Comparative studies should include multiple ERFs to understand their distinct and overlapping roles. For example, ERF1, ORA59, and ERF96 all bind to GCC box elements in defense gene promoters but may have different activation potentials and target specificities .

What techniques can be used to study ERF120 binding to target gene promoters?

To investigate ERF120's DNA binding properties and transcriptional regulation activities:

• Chromatin Immunoprecipitation (ChIP):

  • Cross-link protein-DNA complexes in plant tissue

  • Immunoprecipitate with ERF120 antibody

  • Analyze bound DNA fragments by qPCR or sequencing

  • Focus on promoters containing GCC box elements (AGCCGCC)

• Electrophoretic Mobility Shift Assay (EMSA):

  • Use recombinant ERF120 or nuclear extracts

  • Test binding to labeled DNA probes containing potential binding sites

  • Compete with unlabeled probes to confirm specificity

  • Super-shift with ERF120 antibody to confirm complex identity

• Transactivation Assays:

  • Create reporter constructs with potential target promoters

  • Co-express with ERF120 in plant protoplasts

  • Measure reporter activity under various conditions

  • Include mutated GCC box controls to confirm binding specificity

Studies with other ERFs show that they predominantly bind to GCC box elements in promoters of defense genes like PDF1.2, PR3, and PR4 .

How can non-specific binding issues with ERF120 antibody be resolved?

When encountering non-specific binding with ERF120 antibody, implement these troubleshooting steps:

• Optimization of blocking conditions:

  • Test different blocking agents (5% BSA, 5% non-fat dry milk, commercial blockers)

  • Extend blocking time (2-3 hours at room temperature or overnight at 4°C)

  • Add 0.1-0.3% Tween-20 to reduce hydrophobic interactions

• Antibody dilution optimization:

  • Test serial dilutions (1:500 to 1:5000) to determine optimal concentration

  • Use an antibody diluent containing 0.05% Tween-20 and 1% blocking agent

  • Consider overnight incubation at 4°C for primary antibody

• Stringent washing:

  • Increase number of washes (5-6 times for 5-10 minutes each)

  • Use higher salt concentration in wash buffer (up to 500 mM NaCl)

  • Add 0.1% SDS to wash buffer for extremely sticky antibodies

• Sample preparation improvements:

  • Include additional protease inhibitors in extraction buffer

  • Pre-clear lysates with Protein A/G beads

  • Consider subcellular fractionation to enrich for nuclear fraction (where ERF120 is expected)

What factors affect ERF120 expression during experimental manipulations?

To account for variables affecting ERF120 expression in experimental designs:

• Hormone treatments:

  • Ethylene (ET) precursor ACC and methyl jasmonate (MeJA) typically induce ERF expression

  • Monitor treatment timing carefully (6-24 hours typically optimal)

  • Note that salicylic acid (SA) may repress some ERF expression

• Developmental stage:

  • ERF expression varies by tissue type and developmental stage

  • Young tissues and vascular tissues often show higher expression

  • Root tips and guard cells may have constitutively higher levels

• Environmental conditions:

  • Light conditions affect ERF expression

  • Temperature stress alters expression patterns

  • Control growth conditions carefully for reproducible results

• Pathogen challenge variables:

  • ERF induction kinetics differ by pathogen type

  • Document the exact timing of tissue collection post-infection

  • Consider dose-dependent responses to pathogen exposure

Research shows that ERF gene expression can be rapidly induced by pathogen challenge and is robust against single hormone pathway disruption .

How should researchers interpret contradictory results in ERF120 expression studies?

When faced with contradictory ERF120 expression data, consider these methodological approaches to resolution:

• Technique-specific variables:

  • Western blot vs. qPCR results may differ due to post-transcriptional regulation

  • Antibody detection may be affected by protein modifications or complex formation

  • Compare results across multiple detection methods

• Biological variables to consider:

  • Growth conditions may significantly affect ERF expression patterns

  • Genetic background differences between Arabidopsis ecotypes

  • Age and developmental stage of plant material

  • Circadian regulation of ERF expression

• Resolution approaches:

  • Use time-course experiments to capture expression dynamics

  • Include positive controls (known induced ERFs) in each experiment

  • Measure multiple outputs (protein levels, target gene expression)

  • Consider tissue-specific analyses rather than whole-plant measurements

Studies of ERF family members show that their expression can be regulated through complex feedback loops and cross-regulation with other transcription factors .

What are the most effective experimental designs for studying ERF120 function in stress responses?

For comprehensive functional characterization of ERF120 in stress responses:

• Genetic manipulation approaches:

  • Generate and characterize erf120 knockout/knockdown lines

  • Create ERF120 overexpression lines

  • Develop inducible expression systems for temporal control

  • Compare phenotypes under normal and stress conditions

• Multi-omics integration:

  • Combine transcriptomics, proteomics, and metabolomics

  • Identify direct and indirect targets through ChIP-seq and RNA-seq

  • Map the stress response network through protein-protein interaction studies

  • Validate key nodes through targeted genetic manipulation

• Physiological measurements:

  • Document morphological responses to stress

  • Measure stress hormones (ethylene, JA, ABA)

  • Quantify stress metabolites

  • Assess pathogen growth in challenged plants

Research on ERF family members indicates they integrate signals from multiple hormone pathways and can affect plant growth, development, and stress responses .

How can protein-protein interactions of ERF120 be effectively studied?

To characterize ERF120's protein interaction network:

• Co-immunoprecipitation (Co-IP):

  • Use ERF120 antibody to pull down native protein complexes

  • Confirm specificity with immunoblotting

  • Identify interacting partners through mass spectrometry

  • Validate key interactions with reciprocal Co-IP

• Yeast two-hybrid screening:

  • Create ERF120 bait constructs (consider domain-specific constructs)

  • Screen against cDNA libraries from relevant tissues/conditions

  • Validate interactions through deletion mapping

  • Confirm in planta through BiFC or Co-IP

• Bimolecular Fluorescence Complementation (BiFC):

  • Generate fusion constructs with split fluorescent protein fragments

  • Co-express in Arabidopsis protoplasts or via transient expression

  • Visualize interaction through fluorescence microscopy

  • Include appropriate negative controls (non-interacting protein pairs)

• Proximity-dependent labeling:

  • Create ERF120-BioID or TurboID fusion proteins

  • Express in plants under native or inducible promoters

  • Identify proximal proteins through streptavidin pulldown and MS

  • Distinguish between direct and proximal interactions