ERF105 Antibody

What is ERF105 and what is its functional role in plants?

ERF105 (ETHYLENE RESPONSE FACTOR 105) is a plant-specific transcription factor belonging to group IXb of the ERF family in Arabidopsis thaliana. It functions as an important regulatory factor in plant stress responses, particularly in freezing tolerance and cold acclimation processes. Research has demonstrated that ERF105 plays a critical role in regulating the expression of cold-responsive genes, suggesting its action is linked to the CBF regulon, a key regulatory pathway in cold stress responses .

Beyond cold stress, ERF105 is also involved in fast retrograde signaling responses and high light acclimation, indicating its broader role in abiotic stress adaptation. The protein contains a highly conserved AP2/ERF domain characteristic of this transcription factor family, which is responsible for DNA binding and transcriptional regulation activities .

How is ERF105 related structurally and functionally to other ERF transcription factors?

ERF105 belongs to a subfamily of four closely related transcription factors in Arabidopsis (ERF102, ERF103, ERF104, and ERF105) that show significant sequence conservation. Comparative analysis of their amino acid sequences reveals approximately 40% sequence identity among all four proteins, with particularly high conservation in the AP2/ERF domain that mediates DNA binding .

The proteins exhibit clear phylogenetic relationships, with ERF104 and ERF105 forming one branch (sharing 52% amino acid identity), while ERF102 and ERF103 form another branch (sharing 67% amino acid identity) on the phylogenetic tree . This structural similarity suggests potential functional redundancy, which is confirmed by their overlapping but not identical roles in various stress responses.

The table below summarizes the structural relationships between these related ERF proteins:

What expression patterns does ERF105 exhibit across different tissues?

Analysis of tissue-specific expression using promoter:GUS reporter lines has revealed that ERF105, along with the other members of its subfamily (ERF102-104), shows predominant expression in root tissues, though with distinct patterns for each family member. While specific ERF105 expression domains were not detailed in the available search results, the related family members show expression in the root meristem (ERF103), quiescent center (ERF104), and root vasculature (all four members including ERF105) .

This tissue-specific expression pattern provides important clues about the potential developmental and physiological roles of ERF105, suggesting functions that may be associated with root development and stress sensing, particularly in the vascular tissues where stress signals are often transmitted throughout the plant.

How does ERF105 respond transcriptionally to different abiotic stresses and hormones?

ERF105 exhibits rapid and often transient transcriptional responses to various plant hormones and abiotic stresses, making it an excellent marker for early stress signaling events. The gene shows remarkable responsiveness to multiple hormones:

• Auxin response: ERF105 transcript abundance increases approximately 130-fold within 30 minutes of auxin (NAA) treatment, followed by a rapid decline to only 2-fold elevation after 2 hours, demonstrating an extremely fast and transient response .

• Salicylic acid (SA) response: ERF105 transcription is downregulated to approximately 50% of initial levels within 2 hours of SA treatment .

• Cold stress response: ERF105 expression is induced by cold temperatures, consistent with its role in freezing tolerance and cold acclimation .

• Other hormones: Previous research has shown that ERF105 is also regulated by ethylene, jasmonate, and abscisic acid, all of which are involved in cold stress responses .

This dynamic transcriptional regulation in response to multiple signals positions ERF105 as an integrative hub in hormone signaling networks during plant stress responses, particularly cold stress adaptation.

What molecular mechanisms underlie ERF105's role in cold stress tolerance?

Research indicates that ERF105 functions as a key regulatory element in plant cold acclimation pathways. The strongly reduced expression of cold-responsive genes in ERF105 mutants during cold acclimation suggests that its regulatory action is connected to the CBF (C-repeat binding factor) regulon, a central pathway in plant cold stress responses .

As a transcription factor, ERF105 likely acts by binding to specific DNA sequences in the promoters of target genes, thereby activating or repressing their expression. The AP2/ERF domain characteristic of this protein family is known to recognize GCC-box elements in promoters, though specific binding sites for ERF105 would require experimental validation using techniques such as chromatin immunoprecipitation (ChIP).

The functional importance of ERF105 in cold stress tolerance is further supported by phenotypic analyses of loss-of-function mutants, which show impaired freezing tolerance and cold acclimation capabilities .

How can researchers effectively use ERF105 antibodies to investigate protein-protein interactions?

When investigating ERF105 protein-protein interactions, researchers should employ co-immunoprecipitation (Co-IP) techniques optimized for plant transcription factors. The procedure should begin with tissue selection based on the expression pattern data, focusing primarily on root tissues where ERF105 shows high expression .

For effective Co-IP experiments:

• Crosslinking: Use formaldehyde (1-1.5%) to crosslink protein complexes in planta before tissue harvesting.

• Extraction buffer optimization: Include protease inhibitors, phosphatase inhibitors, and mild detergents (0.1% NP-40) to maintain protein interactions while effectively lysing cells.

• Pre-clearing: Pre-clear lysates with protein A/G beads to reduce non-specific binding.

• Antibody selection: Use a validated anti-ERF105 antibody such as the CSB-PA837773XA01DOA antibody designed for Arabidopsis thaliana .

• Controls: Include appropriate negative controls (non-immune IgG, tissue from erf105 knockout plants) and positive controls (known interaction partners if available).

• Verification: Confirm interactions through reciprocal Co-IP and additional techniques such as yeast two-hybrid or bimolecular fluorescence complementation.

The identification of interaction partners can provide crucial insights into how ERF105 functions within larger transcriptional complexes to regulate gene expression during stress responses.

What are the optimal methods for validating ERF105 antibody specificity?

Validating ERF105 antibody specificity is crucial for reliable experimental results, particularly given the high sequence similarity between ERF105 and its close relatives (ERF102-104). A comprehensive validation approach should include:

For researchers working with commercially available antibodies like CSB-PA837773XA01DOA , requesting validation data from the manufacturer and performing independent validation experiments is strongly recommended.

What protocols should be followed for ChIP-seq experiments using ERF105 antibodies?

Chromatin immunoprecipitation followed by sequencing (ChIP-seq) is a powerful technique to identify genome-wide binding sites of ERF105. For optimal results with ERF105 antibodies, researchers should follow this specialized protocol:

• Plant material selection: Choose tissues with high ERF105 expression, primarily root tissues . Consider using plants exposed to cold or other stresses that induce ERF105 expression.

• Crosslinking: Crosslink plant tissue with 1% formaldehyde for 10 minutes under vacuum, followed by quenching with 0.125M glycine.

• Chromatin extraction and fragmentation: Extract chromatin in a buffer containing protease inhibitors and fragment using sonication to achieve fragments of 200-500 bp.

• Antibody selection: Use a validated anti-ERF105 antibody like CSB-PA837773XA01DOA , but ensure it has been validated for ChIP applications specifically.

• Pre-clearing and immunoprecipitation: Pre-clear chromatin with protein A/G beads, then immunoprecipitate with the ERF105 antibody overnight at 4°C.

• Washing and elution: Perform stringent washing steps to remove non-specific interactions, then elute and reverse crosslinks.

• Library preparation: Prepare sequencing libraries using standard protocols, considering the typically low yield of ChIP material from plant samples.

• Bioinformatic analysis: Analyze sequencing data using peak-calling algorithms optimized for transcription factors, and annotate peaks relative to gene models.

• Motif discovery: Perform de novo motif discovery to identify the consensus binding sequence for ERF105, which likely includes GCC-box elements common to ERF transcription factors.

• Validation: Validate selected binding sites using ChIP-qPCR on independent biological replicates.

This protocol should enable identification of ERF105 target genes, providing insights into its regulatory network during stress responses.

How can researchers optimize immunohistochemistry protocols for ERF105 localization studies?

For immunohistochemical detection of ERF105 in plant tissues, researchers need to optimize several parameters to overcome challenges specific to plant samples:

• Tissue fixation: Use a combination of 4% paraformaldehyde with 0.1-0.2% glutaraldehyde to preserve protein antigenicity while maintaining tissue structure.

• Cell wall permeabilization: Incorporate an enzymatic digestion step using a mixture of cellulase (1-2%) and macerozyme (0.2-0.5%) to improve antibody penetration through plant cell walls.

• Antigen retrieval: Perform heat-induced epitope retrieval in citrate buffer (pH 6.0) to expose potentially masked epitopes.

• Blocking: Use a mixture of 5% normal serum, 3% BSA, and 0.1% Triton X-100 in PBS to reduce non-specific binding.

• Antibody incubation: Dilute primary ERF105 antibody (e.g., CSB-PA837773XA01DOA ) optimally (typically 1:100 to 1:500) and incubate for 16-24 hours at 4°C.

• Detection system: Use a fluorescently-labeled secondary antibody system for better signal-to-noise ratio and compatibility with co-localization studies.

• Counterstaining: Include DAPI staining to visualize nuclei, as ERF105 is expected to show nuclear localization based on studies with GFP-ERF fusion proteins .

• Controls: Include essential controls:

  • Negative control: Sections from erf105 knockout plants

  • Absorption control: Antibody pre-incubated with recombinant ERF105 protein

  • Secondary antibody-only control

• Confocal microscopy: Utilize confocal microscopy for high-resolution imaging of ERF105 localization, with special attention to nuclear localization patterns.

This protocol should allow researchers to visualize the tissue-specific and subcellular localization of ERF105, complementing the promoter:GUS studies that have revealed expression patterns of the ERF102-105 gene family .

What specialized equipment is needed for ERF105 antibody-based research?

Research utilizing ERF105 antibodies requires several specialized pieces of equipment to ensure optimal results across different experimental applications:

• Protein electrophoresis and Western blotting system: A high-resolution gel electrophoresis system with semi-dry or wet transfer capabilities is essential for antibody validation and protein detection experiments.

• Fluorescence/chemiluminescence imaging system: A sensitive imaging system with appropriate filters for detecting secondary antibody labels in Western blots and immunohistochemistry applications.

• Controlled environment chambers: Given ERF105's role in cold stress responses, temperature-controlled growth chambers capable of precise temperature regulation are necessary for stress treatment experiments.

• Confocal microscope: For immunolocalization studies, a confocal microscope with appropriate lasers and filters for detecting fluorescently-labeled antibodies and counterstains is required.

• qPCR system: For validating ChIP experiments and monitoring ERF105 gene expression under different conditions.

• Tissue homogenizer: A high-efficiency homogenizer suitable for plant tissues, which can be challenging to disrupt due to cell wall components.

• Ultracentrifuge: For subcellular fractionation studies to examine ERF105 localization biochemically.

• Next-generation sequencing platform: Access to sequencing technology is essential for ChIP-seq experiments to identify genome-wide binding sites.

Proper maintenance and calibration of this equipment are crucial for generating reliable and reproducible data in ERF105 research.

What reference materials should researchers use when working with ERF105 antibodies?

When working with ERF105 antibodies, researchers should utilize the following reference materials to ensure experimental validity and reproducibility:

• Positive control materials:

  • Recombinant ERF105 protein for antibody validation

  • Arabidopsis lines overexpressing ERF105 (35S:ERF lines mentioned in the literature)

  • Known target genes or proteins for functional validation

• Negative control materials:

  • erf105 knockout or knockdown lines for specificity testing

  • Non-immune IgG of the same species as the primary antibody

  • Samples treated with blocking peptides

• Reference sequences:

  • UniProt entry Q8VY90 for Arabidopsis thaliana ERF105 protein sequence

  • TAIR (The Arabidopsis Information Resource) gene model for AT5G51190 (ERF105)

  • Sequence alignments of ERF102-105 to identify unique regions for antibody targeting

• Standard protocols:

  • Plant-specific protocols for protein extraction that account for interfering compounds

  • Optimized immunoprecipitation protocols for plant transcription factors

  • Validated ChIP protocols for Arabidopsis transcription factors

• Antibody validation data:

  • Manufacturer's validation data for commercial antibodies like CSB-PA837773XA01DOA

  • Published literature using the same or similar antibodies

  • Internal validation results documented according to standard reporting guidelines

These reference materials provide critical quality control checks and comparative standards for interpreting experimental results with ERF105 antibodies.

How should researchers design experiments to study ERF105 function in stress responses?

When designing experiments to investigate ERF105 function in stress responses, researchers should implement a comprehensive experimental design that accounts for the complex and dynamic nature of plant stress responses:

• Genetic materials:

  • Include multiple genetic tools: wild-type plants, erf105 single mutants, combined mutants of related genes (erf102/erf103/erf104/erf105), and overexpression lines

  • Consider generating inducible expression lines to control timing of ERF105 expression

  • Include reporter lines (such as promoter:GUS) to monitor expression patterns

• Stress treatment design:

  • Apply relevant stresses incrementally: Use gradual temperature decreases for cold stress rather than sudden shocks

  • Implement time-course experiments to capture both early (30 min, 2 hours) and late responses based on known rapid transcriptional responses of ERF105

  • Consider combinatorial stresses that reflect natural conditions (e.g., cold + drought)

• Molecular analyses:

  • Perform transcriptomic profiling (RNA-seq) comparing wild-type and mutant responses to stress

  • Use ChIP-seq with ERF105 antibodies to identify direct target genes

  • Implement proteomic approaches to identify stress-responsive protein interactions

• Physiological measurements:

  • Quantify relevant physiological parameters (e.g., freezing tolerance, electrolyte leakage, photosynthetic efficiency)

  • Measure specific metabolites associated with cold acclimation

  • Analyze growth parameters under control and stress conditions

• Statistical considerations:

  • Ensure adequate biological replication (minimum n=4 for most experiments)

  • Use appropriate statistical tests for time-course data (repeated measures ANOVA)

  • Implement multivariate analyses to integrate different data types

• Controls and validation:

  • Include appropriate controls for each experimental variable

  • Validate key findings through independent methods

  • Test findings across different developmental stages and environmental conditions

This comprehensive approach will allow researchers to develop a holistic understanding of ERF105 function in plant stress response networks.

What are common challenges in ERF105 antibody experiments and how can they be resolved?

Researchers working with ERF105 antibodies may encounter several common challenges that can be addressed through specific troubleshooting strategies:

• Cross-reactivity with related ERF proteins:

  • Problem: The high sequence similarity (40-67%) between ERF102-105 proteins can lead to antibody cross-reactivity.

  • Solution: Use epitope-specific antibodies targeting unique regions of ERF105; validate specificity using knockout lines for each ERF; perform Western blots on recombinant proteins of all four ERFs to quantify cross-reactivity.

• Low signal in Western blots:

  • Problem: Low abundance of ERF105 in some tissues or conditions.

  • Solution: Enrich nuclear fractions before Western blotting; use plants treated with stress conditions known to upregulate ERF105 (e.g., cold treatment) ; increase protein loading; use enhanced chemiluminescence detection systems; consider immunoprecipitation before Western blotting.

• High background in immunohistochemistry:

  • Problem: Non-specific binding in plant tissues.

  • Solution: Optimize blocking conditions (try different blockers like milk, BSA, or fish gelatin); increase washing stringency; use monovalent antibody fragments; pre-absorb antibody with plant extract from erf105 knockout plants.

• Poor ChIP efficiency:

  • Problem: Low enrichment in ChIP experiments.

  • Solution: Optimize crosslinking conditions; increase antibody amount; verify antibody capacity for recognizing fixed epitopes; consider dual crosslinking with formaldehyde and protein-specific crosslinkers; use plants overexpressing ERF105 for initial protocol optimization.

• Inconsistent results between experiments:

  • Problem: Variable antibody performance across experiments.

  • Solution: Standardize all experimental conditions; aliquot and store antibodies properly to avoid freeze-thaw cycles; validate each new antibody lot; include consistent positive controls in every experiment.

• Difficulty detecting stress-induced changes in ERF105:

  • Problem: Rapid and transient nature of ERF105 responses may lead to missed detection.

  • Solution: Implement detailed time-course experiments with early time points (15, 30, 60 minutes after stress application); consider using phospho-specific antibodies if post-translational modifications affect ERF105 during stress.

Implementing these troubleshooting strategies can significantly improve the reliability and reproducibility of ERF105 antibody-based experiments.