ERF098 Antibody

What is ERF098/TDR1 and what is its function in plants?

ERF098, also known as TDR1 (TRANSCRIPTIONAL REGULATOR OF DEFENSE RESPONSE 1), is a member of the ERF (ethylene response factor) subfamily B-3 of the ERF/AP2 transcription factor family in plants. The protein functions as a transcriptional regulator involved in defense responses and potentially in stress signaling pathways. ERF098 contains one AP2 domain, which is characteristic of this transcription factor family . Similar to other ERFs like ERF8, ERF098 likely plays roles in both developmental processes and adaptation to biotic or abiotic stresses, such as pathogen defense, temperature stress, or drought responses . The ERF subfamily represents one of the largest plant-specific transcription factor families, with 65 ERFs constituting the largest subfamily of the AP2/EREBP family in Arabidopsis .

How does ERF098 relate to other members of the ERF family?

ERF098 belongs to a specific subgroup of ERFs that function in various plant stress responses. While ERF098 has its unique functions, it shares structural and functional similarities with other ERFs. For instance, ERF95-ERF98 are characterized as relatively small ERFs containing 128-139 amino acids . Other ERFs such as ERF8 have been shown to interact with mitogen-activated protein kinases (MPKs), particularly MPK4 and MPK11, which are activated during pathogen-associated molecular pattern recognition . Similar interactions might exist for ERF098, though specific studies would be needed to confirm this. Understanding these relationships is crucial when interpreting experimental results involving multiple ERF family members, as functional redundancy may exist.

What are the primary research applications for ERF098 antibody?

ERF098 antibody serves multiple research purposes in plant molecular biology. The primary applications include:

• Protein Detection: Western blot analysis to quantify ERF098 expression levels across different tissues, developmental stages, or stress conditions.

• Localization Studies: Immunohistochemistry and immunofluorescence to determine the subcellular and tissue localization of ERF098.

• Protein-Protein Interaction Studies: Immunoprecipitation to identify protein complexes involving ERF098.

• Chromatin Immunoprecipitation (ChIP): To identify DNA binding sites and target genes of ERF098.

The antibody enables researchers to track changes in ERF098 expression and activity, which is particularly valuable when studying plant stress responses, similar to how other ERF family members have been studied in pathogen defense and heat stress responses .

How can ERF098 antibody be used to study plant stress responses?

To effectively study plant stress responses using ERF098 antibody, researchers should implement a systematic approach:

• Experimental Design: Set up stress treatments (pathogen exposure, temperature stress, drought) alongside appropriate controls.

• Tissue Sampling: Collect plant tissues at various time points after stress exposure.

• Protein Extraction: Use optimized buffers that preserve post-translational modifications, as phosphorylation may be crucial for ERF activity (as seen with ERF8) .

• Western Blot Analysis: Quantify ERF098 protein levels using the antibody, comparing stressed vs. control conditions.

• Nuclear Fraction Enrichment: Since ERF098 is a transcription factor, enriching nuclear fractions can improve detection sensitivity.

• Correlation Analysis: Compare ERF098 levels with expression of potential target genes to establish functional relationships.

This methodological framework allows researchers to determine whether ERF098 is upregulated, downregulated, or post-translationally modified in response to specific stresses, similar to how ERF95 and ERF97 have been shown to regulate heat stress responses .

What are the optimal conditions for storing and handling ERF098 antibody?

For maximum retention of activity and specificity with ERF098 antibody, researchers should follow these storage and handling protocols:

• Storage: Store the lyophilized antibody in a manual defrost freezer and avoid repeated freeze-thaw cycles which can degrade antibody quality .

• Shipping Conditions: The product is typically shipped at 4°C and should be stored immediately at the recommended temperature upon receipt .

• Reconstitution: When reconstituting lyophilized antibody, use sterile techniques and appropriate buffer systems (typically PBS with 0.1% BSA).

• Working Aliquots: Prepare small working aliquots to minimize freeze-thaw cycles.

• Temperature Management: Keep the antibody on ice during experiments.

• Contamination Prevention: Use clean pipette tips and containers to prevent contamination.

These precautions help maintain antibody specificity and sensitivity, critical for detecting ERF098 which may be expressed at relatively low levels in certain tissues or conditions.

What are the recommended protocols for Western blot detection of ERF098?

For optimal Western blot detection of ERF098, follow this detailed protocol:

• Sample Preparation:

  • Extract proteins using a buffer containing phosphatase inhibitors if phosphorylation status is important (as demonstrated for ERF8) .

  • Include protease inhibitors to prevent degradation.

• Gel Electrophoresis:

  • Use 10-12% SDS-PAGE gels for optimal separation of the ~15-20 kDa ERF098 protein.

  • Load 20-50 μg of total protein per lane.

• Transfer:

  • Use PVDF membrane for better protein retention.

  • Transfer at low voltage (30V) overnight at 4°C for efficient transfer of small proteins.

• Blocking:

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

• Primary Antibody Incubation:

  • Dilute ERF098 antibody in blocking solution (optimal dilution should be determined empirically).

  • Incubate overnight at 4°C with gentle agitation.

• Washing and Secondary Antibody:

  • Wash 3-4 times with TBST.

  • Incubate with HRP-conjugated secondary antibody for 1 hour at room temperature.

• Detection:

  • Use enhanced chemiluminescence detection.

  • Include positive controls and molecular weight markers.

• Controls:

  • Include samples from ERF098 overexpression lines as positive controls.

  • Consider using ERF098 knockout/knockdown lines as negative controls, similar to the approach used with erf8-1 knockdown lines .

This protocol incorporates lessons from studies on related ERF proteins while addressing the specific requirements for detecting ERF098.

How can researchers address non-specific binding issues with ERF098 antibody?

When encountering non-specific binding with ERF098 antibody, implement these troubleshooting strategies:

• Optimize Blocking Conditions:

  • Test different blocking agents (milk, BSA, commercial blocking buffers).

  • Increase blocking time or concentration.

• Antibody Dilution Series:

  • Perform a dilution series to determine optimal antibody concentration.

  • Consider using a 1:1000 to 1:5000 range for primary antibody.

• Wash Buffer Modifications:

  • Increase Tween-20 concentration in wash buffer to 0.1-0.3%.

  • Add 0.1% SDS to reduce non-specific hydrophobic interactions.

• Pre-absorption:

  • Pre-absorb the antibody with proteins from knockout/knockdown plant tissues.

  • This removes antibodies that bind to proteins other than ERF098.

• Epitope Competition:

  • If the immunogen is available, perform competition assays by pre-incubating the antibody with the immunogen peptide.

• Cross-Reactivity Analysis:

  • Test the antibody on tissues from plants overexpressing different ERF family members to assess cross-reactivity, similar to verification methods used for other ERF antibodies .

• Sample Preparation Refinement:

  • Enrich nuclear fractions where ERF098 is predominantly located.

  • Use phosphatase inhibitors if phosphorylation affects binding specificity.

These approaches systematically address the common causes of non-specific binding, improving the reliability of ERF098 detection in experimental settings.

How should researchers validate ERF098 antibody specificity?

To rigorously validate ERF098 antibody specificity, employ multiple complementary approaches:

• Genetic Controls:

  • Test the antibody on samples from ERF098 knockout/knockdown lines, which should show reduced or absent signal.

  • Use ERF098 overexpression lines as positive controls, similar to the DEX-inducible ERF8 overexpression system .

• Western Blot Analysis:

  • Verify that the detected band appears at the expected molecular weight (~15-20 kDa).

  • Confirm signal reduction in knockdown lines and signal increase in overexpression lines.

• Immunoprecipitation-Mass Spectrometry:

  • Perform immunoprecipitation followed by mass spectrometry to confirm that the antibody is capturing ERF098.

  • This approach can also identify potential interacting partners.

• Epitope Competition Assay:

  • Pre-incubate the antibody with excess immunizing peptide before immunoblotting.

  • Signal should be significantly reduced if the antibody is specific.

• Cross-Reactivity Assessment:

  • Test against closely related ERF proteins (ERF95-97) to ensure specificity within the family.

  • This is especially important given the high sequence similarity among ERF family members .

• Phosphorylation-Dependent Recognition:

  • If ERF098 is phosphorylated similar to ERF8 , test antibody recognition of both phosphorylated and non-phosphorylated forms.

This multi-faceted validation approach ensures confidence in experimental results and interpretations when using ERF098 antibody.

How can ERF098 antibody be used in chromatin immunoprecipitation (ChIP) experiments?

For successful ChIP experiments with ERF098 antibody, follow this specialized protocol:

• Crosslinking:

  • Crosslink plant tissues with 1% formaldehyde for 10-15 minutes under vacuum.

  • Quench with 0.125 M glycine for 5 minutes.

• Tissue Processing:

  • Grind tissues in liquid nitrogen to a fine powder.

  • Suspend in ChIP extraction buffer with protease inhibitors.

• Chromatin Shearing:

  • Sonicate to generate 200-500 bp DNA fragments.

  • Verify fragment size by agarose gel electrophoresis.

• Immunoprecipitation:

  • Pre-clear chromatin with protein A/G beads.

  • Incubate cleared chromatin with ERF098 antibody overnight at 4°C.

  • Include IgG control and input samples.

• Washing and DNA Recovery:

  • Wash immunoprecipitated complexes with increasingly stringent buffers.

  • Reverse crosslinks and purify DNA.

• qPCR Analysis:

  • Design primers for potential ERF098 binding sites, which likely include GCC-box elements similar to other ERFs.

  • Normalize to input and IgG controls.

• Next-Generation Sequencing:

  • For genome-wide binding site identification, prepare ChIP-seq libraries.

  • Analyze using bioinformatics pipelines to identify enriched motifs.

• Validation Approaches:

  • Confirm binding sites using electrophoretic mobility shift assays (EMSA).

  • Perform reporter gene assays to verify functional significance.

This approach enables identification of ERF098 target genes, similar to how genome-wide transcriptomic analysis has revealed ERF8's role in regulating genes involved in pathogen defense and cell death regulation .

What approaches can be used to study ERF098 phosphorylation and its impact on function?

To investigate ERF098 phosphorylation and its functional implications, implement this methodological framework:

• Phosphorylation Site Prediction:

  • Use bioinformatics tools to predict potential phosphorylation sites based on conserved MAP kinase phosphorylation motifs (S/T-P), similar to the approach used for ERF8 .

• In Vitro Kinase Assays:

  • Express and purify recombinant ERF098 protein.

  • Perform in vitro kinase assays with candidate kinases (e.g., MPK4, MPK11) that have been shown to phosphorylate other ERFs .

  • Analyze phosphorylation using autoradiography or phospho-specific antibodies.

• Mass Spectrometry Analysis:

  • Perform LC-MS/MS phosphopeptide analysis after in vitro phosphorylation.

  • This technique can identify specific phosphorylation sites, as demonstrated with ERF8 .

• Phosphomimetic and Phospho-dead Mutants:

  • Generate site-directed mutants (S/T→A for phospho-dead; S/T→D/E for phosphomimetic).

  • Test these mutants in functional assays to determine how phosphorylation affects:

    • DNA binding affinity

    • Protein stability

    • Transcriptional activity

    • Protein-protein interactions

• Phosphorylation-Specific Antibodies:

  • Develop antibodies that specifically recognize phosphorylated forms of ERF098.

  • Use these to track phosphorylation status under different stress conditions.

• In Vivo Phosphorylation Studies:

  • Treat plants with stress stimuli known to activate MAPKs.

  • Immunoprecipitate ERF098 and analyze phosphorylation status.

  • Use phosphatase treatments as controls.

This comprehensive approach allows researchers to determine whether ERF098, like ERF8, is regulated by phosphorylation and how this modification affects its function in plant stress responses .

How does ERF098 function compare to other ERF family members in stress response pathways?

ERF098's role in stress response pathways can be understood through careful comparative analysis with other ERF family members:

• Functional Redundancy vs. Specificity:

  • While single mutants of ERF family members like ERF95 and ERF97 show wild-type phenotypes in stress responses, double or quadruple mutants may reveal phenotypes due to functional redundancy .

  • Researchers should consider generating ERF098 mutants in combination with closely related ERFs to fully uncover its function.

• Stress-Specific Responses:

  • Different ERFs respond to distinct stresses: some ERFs like ERF95/97 are primarily involved in heat stress responses , while others like ERF8 function in pathogen defense .

  • Study ERF098 under multiple stress conditions to determine its specific role.

• Signaling Pathway Integration:

  • ERF8 integrates ABA signaling and immune responses , serving as a node of crosstalk between abiotic and biotic stress responses.

  • Investigate whether ERF098 similarly integrates multiple signaling pathways.

• Transcriptional Regulation Mechanisms:

  • Some ERFs function as transcriptional repressors through EAR motifs (like ERF8) , while others act as activators.

  • Analyze ERF098 sequence for repression/activation domains to predict its transcriptional activity.

• Experimental Approach:

  • Conduct RNA-Seq with ERF098 overexpression or knockout lines under different stress conditions.

  • Compare differentially expressed genes with those regulated by other ERFs to identify unique and shared targets.

This comparative analysis provides a framework for positioning ERF098 within the complex network of plant stress responses, guiding more targeted research questions.

What advanced methodologies can be used to study ERF098 protein-protein interactions?

To comprehensively characterize ERF098 protein-protein interactions, employ these advanced methodological approaches:

• Co-Immunoprecipitation (Co-IP):

  • Use ERF098 antibody to precipitate native protein complexes from plant tissues.

  • Identify interacting partners by mass spectrometry.

  • Include appropriate controls (IgG, knockout lines) to filter non-specific interactions.

  • This approach has successfully identified MAP kinase interactions with ERF8 .

• Yeast Two-Hybrid (Y2H) Screening:

  • Use ERF098 as bait to screen plant cDNA libraries.

  • Confirm interactions with targeted Y2H assays against specific candidate proteins.

  • Consider domain-specific constructs to map interaction surfaces.

• Bimolecular Fluorescence Complementation (BiFC):

  • Fuse ERF098 and candidate interactors to complementary fragments of fluorescent proteins.

  • Express in plant cells to visualize interactions in vivo.

  • This technique provides spatial information about where interactions occur subcellularly.

• Förster Resonance Energy Transfer (FRET):

  • Tag ERF098 and interacting proteins with appropriate fluorophore pairs.

  • Measure energy transfer to quantitatively assess interactions.

  • This technique allows real-time monitoring of dynamic interactions.

• Proximity-Dependent Biotin Identification (BioID):

  • Fuse ERF098 to a promiscuous biotin ligase.

  • Express in plants and identify biotinylated proteins by mass spectrometry.

  • This method captures both stable and transient interactions in their native cellular context.

• Single-Chain Variable Fragment (scFv) Applications:

  • Develop scFv constructs against ERF098, similar to approaches used in antibody structural studies .

  • These can be used as research tools to study protein-protein interactions without disrupting native complexes.

• Cryo-Electron Microscopy (Cryo-EM):

  • For structural characterization of stable complexes.

  • Address preferred orientation issues by using techniques like scFv constructs instead of larger antibody formats .

These methodologies provide complementary information about ERF098's interaction network, helping to elucidate its role in transcriptional regulation and stress response pathways.