ERF037 Antibody

What is ERF037 and why is it important in plant research?

ERF037 (AT1G77200) is a transcription factor belonging to the Ethylene Response Factor (ERF) family in Arabidopsis thaliana, which regulates genes involved in plant responses to environmental stresses and developmental cues. As a member of the AP2/ERF transcription factor superfamily, ERF037 contains a conserved DNA-binding domain that recognizes GCC-box elements in promoters of stress-responsive genes. The study of ERF037 is particularly valuable for understanding plant molecular responses to biotic and abiotic stresses, as these transcription factors act as critical regulatory nodes in stress signaling networks. Antibodies against ERF037 enable researchers to investigate protein accumulation patterns, subcellular localization, protein-protein interactions, and post-translational modifications that affect ERF037 function. These investigations can provide crucial insights into the molecular mechanisms underlying plant stress responses and potential applications in improving crop resilience .

What are the key specifications of commercially available ERF037 antibodies?

The ERF037 antibody available for plant research is typically a rabbit polyclonal antibody purified through antigen affinity chromatography, providing high specificity for the target protein. These antibodies are generally supplied unconjugated, allowing researchers flexibility in choosing detection systems appropriate for their specific experimental needs. The immunogen used for antibody production is recombinant Arabidopsis thaliana ERF037 protein, ensuring recognition of plant-specific epitopes. According to supplier specifications, the antibody has been validated for ELISA and Western blotting applications, with the latter being particularly useful for assessing ERF037 protein levels in plant tissues under different experimental conditions. The standard product size is approximately 2mg of purified antibody, accompanied by 200μg of antigen (which can serve as a positive control) and 1ml of pre-immune serum (functioning as a negative control) to facilitate experimental validation and troubleshooting .

How should ERF037 antibody be stored and handled to maintain optimal activity?

To preserve the functional integrity of ERF037 antibody, proper storage and handling practices are essential for maintaining its specificity and sensitivity in experimental applications. The antibody should be stored at either -20°C or -80°C for long-term preservation, with -80°C being preferable for extended storage periods beyond six months. When working with the antibody, researchers should aliquot the stock solution into smaller volumes upon receipt to minimize freeze-thaw cycles, as repeated freezing and thawing can lead to protein denaturation and diminished antibody performance. For short-term use, antibody aliquots can be kept at 4°C for up to one week, but should not be left at room temperature for extended periods. When preparing working dilutions, it is advisable to use fresh buffer systems that include protein stabilizers such as BSA (1-5%) to prevent non-specific adsorption to tube walls and maintain antibody activity. Additionally, all solutions that come into contact with the antibody should be prepared with high-quality, nuclease-free water to prevent contamination that could affect experimental outcomes .

How should I design Western blot experiments to detect ERF037 in plant samples?

When designing Western blot experiments to detect ERF037 in plant tissues, several methodological considerations will significantly impact success and reproducibility. Begin by optimizing protein extraction using a buffer system containing protease inhibitors, reducing agents, and phosphatase inhibitors if investigating post-translational modifications. For Arabidopsis samples, a buffer containing 50mM Tris-HCl (pH 7.5), 150mM NaCl, 1% Triton X-100, 1mM DTT, and protease inhibitor cocktail typically yields good results. Load 20-40μg of total protein per lane, as transcription factors like ERF037 are often expressed at relatively low levels. Use 10-12% SDS-PAGE gels for optimal resolution, as ERF037 has a molecular weight of approximately 25-30 kDa. Following transfer to PVDF membranes (which generally perform better than nitrocellulose for plant proteins), block with 5% non-fat dry milk or 3% BSA in TBS-T for 1 hour at room temperature. For primary antibody incubation, a dilution range of 1:500 to 1:2000 is recommended for initial optimization, with overnight incubation at 4°C to maximize specific binding while minimizing background. Include both positive controls (recombinant ERF037 protein provided with the antibody) and negative controls (pre-immune serum) to validate specificity .

What are the recommended procedures for immunoprecipitation of ERF037 from plant extracts?

Immunoprecipitation (IP) of ERF037 from plant tissues requires careful optimization to overcome challenges associated with low abundance of transcription factors and potential cross-reactivity issues. Begin with 500-1000 mg of fresh plant tissue and extract proteins using a non-denaturing buffer (50mM Tris-HCl pH 7.5, 150mM NaCl, 0.5% NP-40, 1mM EDTA, 3mM DTT, and protease inhibitor cocktail) that preserves protein-protein interactions. Pre-clear the lysate with 50μl of Protein A/G agarose beads for 1 hour at 4°C to reduce non-specific binding. For the IP reaction, use 2-5μg of ERF037 antibody per mg of total protein extract, and incubate overnight at 4°C with gentle rotation to maximize antigen-antibody binding. Capture the immune complexes using 50μl of pre-equilibrated Protein A agarose beads (for rabbit polyclonal antibodies) for 3 hours at 4°C. Perform thorough washing steps (at least 4-5 washes) with decreasing salt concentrations to remove non-specifically bound proteins while preserving specific interactions. When eluting the immunoprecipitated proteins, use a gentle approach with either low pH elution buffer (0.1M glycine, pH 2.5) followed by immediate neutralization, or SDS sample buffer heated to 95°C for 5 minutes. Validate IP success through Western blotting using a portion of the eluate, and consider performing reverse IP experiments to confirm specificity of any protein-protein interactions identified .

How can I optimize immunohistochemistry protocols for ERF037 localization in plant tissues?

Optimizing immunohistochemistry (IHC) protocols for ERF037 localization in plant tissues requires attention to several critical parameters that influence specificity, sensitivity, and tissue morphology preservation. Start with freshly harvested plant tissues and fix immediately in 4% paraformaldehyde in PBS (pH 7.4) for 12-16 hours at 4°C, which preserves protein antigenic sites while maintaining tissue architecture. For woody tissues, vacuum infiltration of the fixative is recommended to ensure complete penetration. After fixation, dehydrate tissues through an ethanol series and embed in either paraffin for thin sectioning (5-7μm) or in a plant-specific resin like LR White for semi-thin sectioning (1-2μm). During the antigen retrieval step, which is crucial for many plant tissues due to cross-linking during fixation, use citrate buffer (10mM, pH 6.0) at 95°C for 20-30 minutes, followed by gradual cooling to room temperature. Block endogenous peroxidase activity with 3% hydrogen peroxide if using HRP-based detection systems, and minimize non-specific binding with 5% normal serum from the same species as the secondary antibody. For primary antibody incubation, dilutions between 1:100 and 1:500 should be tested, with overnight incubation at 4°C in a humidified chamber to prevent tissue drying. Include appropriate controls, including sections incubated with pre-immune serum and absorption controls using the immunizing peptide, to validate staining specificity .

What are common causes of weak or absent signals when using ERF037 antibody in Western blots?

When experiencing weak or absent signals in Western blots with ERF037 antibody, several technical and biological factors may be contributing to the problem. Insufficient protein extraction is a common issue when working with plant transcription factors like ERF037, which are often expressed at low levels and may be localized in the nucleus, requiring more rigorous extraction methods. Try incorporating sonication steps (3-5 pulses of 10 seconds each) during extraction or using specialized nuclear protein extraction kits to improve yield. Protein degradation during extraction can be addressed by working quickly at 4°C and increasing the concentration of protease inhibitors, particularly serine and cysteine protease inhibitors that are common in plant tissues. Inefficient transfer of proteins to membranes may occur with transcription factors; optimize transfer conditions by using wet transfer systems at lower voltages (30V) for longer periods (overnight) with the addition of 0.1% SDS to the transfer buffer to facilitate migration of nuclear proteins. Antibody concentration may need adjustment; if using a 1:1000 dilution yields weak signals, try more concentrated solutions (1:500 or even 1:250) with longer incubation times. Finally, detection sensitivity can be enhanced by switching from colorimetric to chemiluminescent or enhanced chemiluminescent (ECL) detection systems, with extended exposure times up to 20-30 minutes for very low abundance proteins .

How can I minimize background issues when performing immunofluorescence with ERF037 antibody?

High background is a frequent challenge in plant immunofluorescence studies using ERF037 antibody, often arising from multiple sources that require systematic troubleshooting. Autofluorescence from plant tissues, particularly chlorophyll, lignin, and phenolic compounds, can be reduced through several approaches: pre-treatment with 0.1% sodium borohydride for 10 minutes, incubation in 0.3M glycine for 15 minutes, or using specific bandpass filters that avoid the chlorophyll emission spectrum (650-750 nm). Non-specific antibody binding can be addressed by extending the blocking step to 2 hours at room temperature using 5% BSA with 0.3% Triton X-100 in PBS, and adding 5% normal serum from the species of the secondary antibody. Overfixation of tissues can mask epitopes; optimize fixation by reducing paraformaldehyde concentration to 2% and fixation time to 8-10 hours, followed by more rigorous antigen retrieval treatments. For plant tissues, enzymatic antigen retrieval using a cocktail of cell wall-degrading enzymes (0.1% cellulase, 0.05% macerozyme, 0.1% pectolyase) for 20 minutes at room temperature often improves antibody accessibility to nuclear proteins. Cross-reactivity with similar ERF family members can be minimized by pre-absorbing the antibody with plant extracts from ERF037 knockout mutants or with recombinant proteins of closely related ERF family members, thereby enhancing specificity for the target protein .

What strategies can improve specificity when using ERF037 antibody in chromatin immunoprecipitation (ChIP) assays?

Chromatin immunoprecipitation (ChIP) using ERF037 antibody presents unique challenges due to the cross-reactivity potential with related ERF family members and the dynamic nature of transcription factor-DNA interactions. To improve specificity in ChIP assays, begin by optimizing crosslinking conditions specifically for plant transcription factors; while 1% formaldehyde for 10 minutes is standard, ERF proteins often benefit from dual crosslinking with 1.5mM EGS (ethylene glycol bis(succinimidyl succinate)) for 30 minutes followed by 1% formaldehyde for 10 minutes, which better preserves transient DNA-protein interactions. Sonication parameters should be carefully calibrated for plant tissues to achieve chromatin fragments of 200-500 bp; typically, 15-20 cycles of 30 seconds on/30 seconds off at medium power works well for Arabidopsis seedlings. Pre-clearing the chromatin with protein A beads conjugated to non-immune IgG for 2 hours significantly reduces non-specific binding. For the immunoprecipitation step, using higher antibody concentrations (5-10 μg per reaction) with longer incubation times (overnight at 4°C) improves capture of low-abundance transcription factors. Implementing stringent washing conditions with increasing salt concentrations (150mM to 500mM NaCl) helps eliminate non-specific interactions. Additionally, conducting parallel ChIP experiments with an unrelated antibody of the same isotype and with antibodies against known ERF family members allows for comparative analysis to distinguish genuine ERF037 binding sites from artifacts or family-wide binding regions. Finally, validation of ChIP results through independent methods such as EMSA (Electrophoretic Mobility Shift Assay) with recombinant ERF037 protein provides strong confirmation of direct binding to identified genomic regions .

How can ERF037 antibody be used for studying protein-protein interactions in stress response pathways?

ERF037 antibody provides powerful tools for investigating protein-protein interactions that mediate transcription factor function in plant stress response networks. Co-immunoprecipitation (Co-IP) followed by mass spectrometry represents the most comprehensive approach for identifying novel interaction partners of ERF037. For this application, crosslinking with protein-interaction preserving agents such as DSP (dithiobis(succinimidyl propionate)) at 1-2mM for 30 minutes prior to cell lysis helps stabilize transient interactions that may occur during stress responses. When performing Co-IP from plants exposed to different stress conditions (e.g., drought, pathogen infection, or ethylene treatment), it is critical to standardize the timing of tissue collection to capture the dynamic changes in interaction networks. The immunoprecipitated complexes should be analyzed using high-sensitivity mass spectrometry with at least three biological replicates per condition to generate statistically robust interaction datasets. For targeted validation of specific interactions, bimolecular fluorescence complementation (BiFC) offers in vivo confirmation, though attention must be paid to the position of the fluorescent protein fragments to avoid steric hindrance of the interaction surfaces. Proximity ligation assay (PLA) provides an alternative validation approach with higher sensitivity, capable of detecting interactions that may be too weak or transient for standard Co-IP techniques. For investigating the influence of post-translational modifications on ERF037 interactions, phospho-specific antibodies can be used in tandem with general ERF037 antibodies to determine how phosphorylation states alter the composition of protein complexes during stress responses .

What approaches can be used to study post-translational modifications of ERF037 using available antibodies?

Post-translational modifications (PTMs) of ERF037 likely play crucial roles in regulating its activity, stability, and interactions during plant stress responses. To comprehensively study these modifications, researchers can employ a multi-faceted approach combining immunoprecipitation with specialized analytical techniques. Begin with large-scale immunoprecipitation using ERF037 antibody from plants subjected to various stress conditions to capture the protein in different modification states. Prior to IP, treat samples with phosphatase inhibitors (50mM NaF, 10mM Na3VO4, 1mM β-glycerophosphate) and deubiquitinase inhibitors (5mM N-ethylmaleimide) to preserve phosphorylation and ubiquitination states respectively. After immunoprecipitation, analyze the purified protein by mass spectrometry using techniques optimized for PTM detection, such as titanium dioxide enrichment for phosphopeptides or antibody-based enrichment for ubiquitinated peptides. For targeted analysis of specific modifications, perform Western blotting on immunoprecipitated ERF037 using modification-specific antibodies such as anti-phosphoserine, anti-phosphothreonine, anti-SUMO, or anti-ubiquitin. Phos-tag SDS-PAGE provides an alternative approach for detecting phosphorylated forms of ERF037 without requiring phospho-specific antibodies. To investigate the functional significance of identified modifications, combine these approaches with site-directed mutagenesis of modified residues in transgenic plants, followed by phenotypic analysis and interaction studies to determine how specific PTMs affect ERF037 function in stress response pathways .

How can ERF037 antibody be employed in high-throughput screening approaches for stress response studies?

High-throughput screening utilizing ERF037 antibody enables systematic investigation of factors affecting this transcription factor's expression, localization, and activity across multiple conditions or genetic backgrounds. Developing ELISA-based quantification methods offers the most straightforward approach for screening large sample sets; optimize a sandwich ELISA using ERF037 antibody as the capture antibody and a biotinylated version as the detection antibody, with standard curves generated using the recombinant protein provided with the antibody kit. This system can be adapted to 384-well formats for screening hundreds of samples from different stress treatments or mutant plant lines. For higher-resolution spatial information across many samples, tissue microarrays (TMAs) prepared from plant samples can be subjected to immunohistochemistry with ERF037 antibody, allowing simultaneous analysis of protein localization across dozens of experimental conditions. Automated immunofluorescence platforms combined with high-content imaging systems can further enhance throughput by capturing subcellular localization data from multiple samples in parallel, with quantitative image analysis software extracting parameters such as nuclear/cytoplasmic distribution ratios. For functional screening approaches, the antibody can be utilized in chromatin immunoprecipitation followed by sequencing (ChIP-seq) across multiple conditions, identifying genome-wide binding patterns that change in response to different stresses or in different genetic backgrounds. Finally, combining these approaches with CRISPR-based genetic screens allows correlation of ERF037 protein levels and localization with phenotypic outcomes, providing comprehensive insights into the functional networks governing plant stress responses .

How should researchers interpret changes in ERF037 protein levels during different stress conditions?

Interpreting changes in ERF037 protein levels during stress responses requires careful consideration of multiple factors to distinguish between direct stress effects and secondary responses. When quantifying ERF037 levels via Western blot or ELISA across stress conditions, researchers should first establish a detailed time course (0, 1, 3, 6, 12, 24, and 48 hours after stress application) to capture both rapid and delayed responses, as transcription factors often show biphasic regulation patterns. Protein level changes should be normalized against multiple reference proteins (such as actin, tubulin, and GAPDH) to account for general effects on protein synthesis or degradation during stress. It is crucial to differentiate between changes in total protein abundance versus subcellular redistribution; a decrease in nuclear fraction accompanied by an increase in cytoplasmic fraction may indicate regulation through nucleo-cytoplasmic shuttling rather than altered expression. Researchers should compare protein-level changes with transcript-level dynamics (measured by RT-qPCR) to determine whether regulation occurs at transcriptional, post-transcriptional, or post-translational levels. The biological significance of observed changes can be assessed by correlation with downstream target gene expression; a modest increase in ERF037 levels may be functionally significant if it coincides with substantial induction of target genes containing GCC-box elements in their promoters. Finally, comparative analysis across related plant species or ecotypes with varying stress tolerance can help distinguish adaptive ERF037 responses from general stress reactions, providing evolutionary context for the observed regulatory patterns .

What are the best approaches for comparing ERF037 binding profiles across different experimental conditions?

Comparing ERF037 binding profiles across different experimental conditions requires robust analytical frameworks that account for technical variability while highlighting biologically meaningful differences. For ChIP-seq experiments examining ERF037 binding under various stress conditions, begin with standardized chromatin preparation and immunoprecipitation protocols that minimize technical variation between samples. Include spike-in normalization using a constant amount of chromatin from a different species (e.g., Drosophila) with a species-specific antibody to provide an internal reference for normalization across samples with potentially different global binding levels. During data analysis, employ both peak-calling approaches to identify discrete binding sites and differential binding analysis to quantify changes in occupancy at specific genomic regions. Analysis should distinguish between changes in binding intensity at constitutive sites versus condition-specific recruitment to new genomic loci, as these patterns may reflect different regulatory mechanisms. Integration of binding data with RNA-seq from matched samples allows correlation between differential binding and transcriptional outcomes, identifying functional binding events. For complex comparisons across multiple conditions, hierarchical clustering or principal component analysis of binding profiles helps identify condition-specific and shared binding patterns. To validate key differential binding events, targeted ChIP-qPCR with biological replicates provides quantitative confirmation of the genome-wide patterns. Finally, motif enrichment analysis comparing the sequences under differential and constitutive binding sites can reveal condition-specific co-factors that might influence ERF037 recruitment to different genomic regions under various stress conditions .

How can contradictory results between antibody-based and tag-based detection of ERF037 be reconciled?

Reconciling contradictory results between antibody-based detection of endogenous ERF037 and detection of tagged versions (such as GFP-ERF037 or HA-ERF037) requires systematic investigation of multiple technical and biological factors that could explain the discrepancies. Begin by validating antibody specificity through Western blot analysis of wild-type plants, ERF037 knockout mutants, and ERF037 overexpression lines, which should demonstrate corresponding absence or elevation of the detected protein band. For tagged protein constructs, verify that the fusion protein maintains functionality through complementation assays in ERF037 mutant backgrounds, as non-functional fusion proteins may exhibit altered stability or localization. Examine the influence of tag position (N-terminal versus C-terminal) on protein behavior, as tags can mask interaction surfaces or localization signals depending on their placement. Compare expression levels between tagged transgenic lines and endogenous protein; artificially high expression from strong promoters can lead to mislocalization or formation of non-physiological protein complexes. Analyze potential differences in post-translational modifications between native and tagged proteins using mass spectrometry, as tags may interfere with certain modifications that affect protein behavior. If discrepancies persist despite these validations, consider the possibility that the antibody recognizes specific conformational states or modified forms of ERF037 that the tagged version may not adopt. In publications, transparently report and discuss these investigations, presenting both antibody-based and tag-based results with appropriate controls to allow readers to evaluate the strengths and limitations of each approach .