ERF069 Antibody

What is ERF069 and what cellular functions does it perform?

ERF069 belongs to the ERF (Ethylene Response Factor) family of transcription factors in Arabidopsis thaliana, which play crucial roles in plant defense responses and stress signaling. These transcription factors typically contain highly conserved ERF domains that bind to GCC-box elements in promoters of target genes, thereby regulating their expression in response to various biotic and abiotic stresses. Based on research on related ERF proteins such as ERF6, ERF069 likely functions downstream of MAPK cascades in pathogen response signaling pathways, potentially regulating defense-related genes in response to fungal pathogens. The protein has been identified with UniProt accession number Q8W4I5, indicating it has been characterized at the sequence level. Similar to other ERFs, it may participate in cross-talk between different hormone signaling pathways, particularly ethylene and jasmonate signaling, which coordinate plant responses to environmental challenges .

What are the recommended applications for ERF069 Antibody?

The ERF069 Antibody has been tested and validated for Enzyme-Linked Immunosorbent Assay (ELISA) and Western Blotting (WB) applications in Arabidopsis thaliana. For Western blotting applications, this antibody allows for specific detection and quantification of ERF069 protein levels under different experimental conditions, facilitating studies on protein expression patterns across developmental stages or in response to various stresses. In ELISA applications, it enables sensitive detection of ERF069 in complex protein mixtures, allowing for quantitative analysis of protein concentration in plant extracts. When designing experiments, researchers should ensure proper identification of the antigen through careful sample preparation and inclusion of appropriate controls. The antibody has undergone affinity purification against the recombinant ERF069 protein, which enhances its specificity for the target protein while minimizing cross-reactivity with other closely related ERF family members .

What are the optimal storage and handling conditions for maintaining ERF069 Antibody activity?

To preserve optimal activity, the ERF069 Antibody should be stored at -20°C or -80°C immediately upon receipt. Refrigeration at 4°C is not recommended for long-term storage as it may result in gradual loss of activity due to protein denaturation and aggregation. Repeated freeze-thaw cycles should be strictly avoided as they can significantly compromise antibody functionality through physical stress on the protein structure. When working with the antibody, aliquoting into smaller volumes before freezing is strongly recommended to prevent unnecessary freeze-thaw cycles. The antibody is supplied in a storage buffer containing 50% glycerol, 0.01M PBS (pH 7.4), and 0.03% Proclin 300 as a preservative, which helps maintain stability during storage. The presence of glycerol prevents freezing at -20°C, maintaining the antibody in a semi-frozen state that protects protein structure. For optimal results, always centrifuge the antibody vial briefly before opening to collect all liquid at the bottom, and avoid contamination by using sterile pipette tips and tubes when handling .

How can ERF069 Antibody be used to study phosphorylation-dependent regulation?

Building on research with related ERF transcription factors, researchers can employ ERF069 Antibody to investigate potential phosphorylation-dependent regulation mechanisms. Experimental approaches should include phosphatase treatment of protein samples prior to Western blotting to identify mobility shifts resulting from phosphorylation states. Researchers can design experiments similar to those used for ERF6, where MPK3/MPK6-mediated phosphorylation was shown to increase protein stability in vivo. For such studies, comparing band patterns between untreated samples and those treated with lambda phosphatase can reveal phosphorylation-dependent mobility shifts. Co-immunoprecipitation experiments using ERF069 Antibody can identify interacting kinases and phosphatases that might regulate ERF069 activity. Additionally, researchers should consider using Phos-tag™ SDS-PAGE, which specifically retards the migration of phosphorylated proteins, allowing better separation and visualization of different phosphorylation states when using the ERF069 Antibody for detection .

What protocols should be followed for immunoprecipitation of ERF069 and its protein complexes?

For effective immunoprecipitation of ERF069 and its associated protein complexes, researchers should follow a methodical approach beginning with optimization of plant tissue extraction conditions. Since ERF069 is a transcription factor, nuclear extraction protocols are recommended using buffers containing 50mM Tris-HCl (pH 7.5), 150mM NaCl, 0.5% NP-40, and protease inhibitor cocktail, supplemented with phosphatase inhibitors if studying phosphorylation states. Pre-clearing the lysate with Protein A/G beads for 1 hour at 4°C will reduce non-specific binding. For the immunoprecipitation, incubate 2-5 μg of ERF069 Antibody with pre-cleared lysate overnight at 4°C with gentle rotation, followed by addition of Protein A beads (as the antibody was raised in rabbit) for 2-3 hours. After thorough washing (at least 4-5 washes with decreasing salt concentrations), elute bound proteins using either low pH glycine buffer (similar to the 0.1M glycine, pH 2.5 method noted in antibody purification protocols) or by boiling in SDS sample buffer. For capturing transient or weak interactions, consider using chemical crosslinking with formaldehyde (1% for 10 minutes) before cell lysis. Analysis of co-immunoprecipitated proteins by mass spectrometry can reveal novel interaction partners of ERF069, potentially identifying components of transcriptional complexes or regulatory proteins .

How can ERF069 Antibody be used in chromatin immunoprecipitation (ChIP) assays?

Adapting the ERF069 Antibody for chromatin immunoprecipitation requires careful optimization to achieve specific enrichment of DNA sequences bound by ERF069 in vivo. Begin with crosslinking using 1% formaldehyde for 10 minutes at room temperature to stabilize protein-DNA interactions, followed by quenching with 0.125M glycine. After nuclear extraction, sonicate chromatin to achieve fragments of 200-500 bp, which can be verified by agarose gel electrophoresis. Pre-clear the chromatin with Protein A beads and normal rabbit IgG before incubation with 5-10 μg of ERF069 Antibody overnight at 4°C. Since this is a polyclonal antibody raised against the full-length recombinant protein, it should recognize multiple epitopes, enhancing ChIP efficiency. Include appropriate controls such as input chromatin (non-immunoprecipitated), mock IP (using normal rabbit IgG), and positive control regions (known targets of related ERF transcription factors). After washing and reverse crosslinking, analyze the enriched DNA by qPCR targeting promoter regions containing GCC-box elements (AGCCGCC), which are common binding sites for ERF transcription factors. For genome-wide binding site identification, the immunoprecipitated DNA can be processed for next-generation sequencing (ChIP-seq), which would reveal the complete set of ERF069 target genes and provide insights into its regulatory network .

What control samples should be included when using ERF069 Antibody for Western blotting?

When conducting Western blotting with ERF069 Antibody, a comprehensive set of controls should be included to ensure reliable and interpretable results. A positive control consisting of recombinant ERF069 protein or lysate from Arabidopsis tissues known to express high levels of ERF069 (such as stressed leaf tissues) should be included to confirm antibody functionality. A negative control using tissue from erf069 knockout or knockdown plants is crucial for verifying antibody specificity. For loading controls, proteins such as actin or GAPDH should be probed on the same membrane or parallel blots to normalize expression levels. Pre-absorption controls, where the antibody is pre-incubated with excess recombinant ERF069 protein before use in Western blotting, can help confirm signal specificity. When studying stress responses, include time-course samples to capture dynamic changes in ERF069 expression, as transcription factors often show transient activation patterns. For phosphorylation studies, include samples treated with phosphatase to identify mobility shifts caused by post-translational modifications, similar to the approach used for ERF6 phosphorylation analysis .

How can potential cross-reactivity with other ERF family members be assessed and addressed?

Due to the high sequence homology among ERF family members in Arabidopsis thaliana, cross-reactivity assessment is crucial when working with ERF069 Antibody. Begin by performing sequence alignment analyses of the ERF069 protein against other ERF family members, particularly focusing on regions that might serve as immunogenic epitopes. Cross-reactivity can be experimentally assessed by testing the antibody against recombinant proteins of closely related ERF members expressed in heterologous systems. Alternatively, use plant tissues from knockout lines of erf069 and closely related erfs to determine if the antibody produces signals in the absence of its intended target. For more precise analysis, consider peptide competition assays where synthetic peptides corresponding to unique regions of ERF069 are used to pre-absorb the antibody before immunoblotting. If cross-reactivity is detected, more stringent washing conditions (higher salt concentration or detergent percentage) may reduce non-specific binding. In cases where cross-reactivity cannot be eliminated, immunoprecipitation followed by mass spectrometry can help identify which specific proteins are being recognized by the antibody .

What factors affect the detection sensitivity of ERF069 using this antibody?

Multiple factors can significantly influence the detection sensitivity of ERF069 using this polyclonal antibody. The expression level of ERF069 is likely stress-inducible, similar to other ERF transcription factors, so timing of sample collection after stress treatment is critical for optimal detection. Based on studies with related ERFs like ERF6, phosphorylation state can affect both protein stability and antibody recognition, potentially requiring phosphatase inhibitors during sample preparation to preserve modification states. The extraction buffer composition greatly impacts recovery of nuclear proteins like ERF069; use buffers containing 0.1-0.5% NP-40 or Triton X-100 with protease inhibitors, and consider including DNA-digesting enzymes to release chromatin-bound transcription factors. Protein transfer efficiency during Western blotting affects detection sensitivity, particularly for transcription factors which may be present at low abundance; consider using PVDF membranes and adding 0.1% SDS to transfer buffer to improve transfer efficiency. The primary antibody concentration should be optimized, typically starting with 1:1000 dilution and adjusting based on signal-to-noise ratio. For enhanced sensitivity in detecting low-abundance ERF069, consider using amplification systems such as biotin-streptavidin or tyramide signal amplification, particularly useful for immunohistochemistry applications .

How can ERF069 Antibody be used in multiplexed immunoassays with other antibodies?

Implementing ERF069 Antibody in multiplexed immunoassays requires strategic planning to overcome potential technical challenges while maximizing information gained from limited samples. For co-immunofluorescence studies, the rabbit-raised ERF069 polyclonal antibody can be paired with mouse-raised antibodies against other proteins of interest, using species-specific secondary antibodies conjugated to different fluorophores with minimal spectral overlap. When designing multiplexed Western blotting, consider the molecular weight of ERF069 (predicted based on amino acid sequence) relative to other proteins of interest to avoid signal overlap. Sequential immunoblotting can be performed by stripping and reprobing membranes, though this may reduce sensitivity with each cycle; alternatively, consider fluorescent Western blotting with differentially labeled secondary antibodies for simultaneous detection of multiple proteins. For co-immunoprecipitation studies investigating ERF069 interactions with other transcription factors or signaling components, a two-step approach may be necessary: first immunoprecipitate with ERF069 Antibody, then probe the precipitated complex with antibodies against suspected interaction partners. In multiplexed ELISA applications, careful titration of both primary and secondary antibodies is essential to minimize cross-reactivity while maintaining sensitivity. For all multiplexed approaches, comprehensive validation using single-antibody controls is crucial to ensure that signal detection and localization are not altered by the presence of multiple antibodies in the system .

How can ERF069 Antibody contribute to understanding plant stress response networks?

The ERF069 Antibody presents a valuable tool for elucidating the role of this transcription factor within the complex signaling networks that regulate plant responses to environmental stresses. By employing this antibody in time-course experiments following exposure to various biotic and abiotic stressors (such as pathogen infection, drought, or temperature extremes), researchers can track the dynamic expression and activation patterns of ERF069. Similar to studies conducted with ERF6, researchers can investigate potential phosphorylation events that may regulate ERF069 activity in response to stress-activated MAPK cascades. The antibody enables identification of upstream regulators through co-immunoprecipitation experiments coupled with mass spectrometry, potentially revealing kinases, phosphatases, or other post-translational modification enzymes that modulate ERF069 function. ChIP-seq experiments utilizing the ERF069 Antibody can map the genome-wide binding sites of this transcription factor under different stress conditions, revealing direct target genes and allowing reconstruction of gene regulatory networks. Integration of these protein-level data with transcriptomic and metabolomic analyses can provide a comprehensive understanding of how ERF069 coordinates molecular responses to environmental challenges, potentially identifying novel stress tolerance mechanisms that could be targeted for crop improvement strategies .

What potential exists for developing ERF069-based molecular tools for crop improvement?

The characterization of ERF069 using the specific antibody could lead to significant advances in developing molecular tools for crop improvement. By establishing the precise role of ERF069 in stress responses through immunoblotting and ChIP studies, researchers can identify whether this transcription factor functions as a positive or negative regulator of stress tolerance pathways. If ERF069 emerges as a positive regulator similar to ERF6, which confers enhanced resistance to fungal pathogens, it could become a candidate for genetic engineering approaches to improve crop resistance to diseases. The antibody enables validation of ERF069 overexpression or modified variants in transgenic plants by confirming proper protein expression and localization. Structure-function studies incorporating site-directed mutagenesis of potential phosphorylation sites (identified based on mobility shifts in Western blots) could lead to development of constitutively active or dominant-negative versions of ERF069 with enhanced regulatory capabilities. For precision breeding approaches, the antibody could help evaluate natural variation in ERF069 expression and modification across different ecotypes or varieties, identifying genetic backgrounds with optimized stress response characteristics. Furthermore, if ERF069 functions as a hub in stress signaling networks, as suggested by studies of related ERFs, it could serve as a target for directed protein engineering or as a scaffold for synthetic transcription factors designed to reprogram plant responses to environmental challenges .