ARF18 Antibody

What is ARF18 and why is it important in plant research?

ARF18 is a transcription factor belonging to the Auxin Response Factor family that mediates auxin-dependent transcriptional regulation. It functions primarily as a transcriptional repressor that binds to specific AuxRR cis-elements in promoters of target genes . ARF18 plays critical roles in multiple developmental processes, including floral organ specification in roses , herbicide resistance in rice , and seed weight determination in rapeseed . Understanding ARF18 function is important for researchers studying plant development, stress responses, and crop improvement, as it represents a key regulatory node in auxin signaling networks that impact agriculturally relevant traits.

What are the specific functions of ARF18 across different plant species?

ARF18 exhibits diverse functions across plant species while maintaining its core role in auxin signaling:

What protein domains characterize ARF18 and how might this affect antibody design?

ARF18 contains three conserved domains that should be considered when designing or selecting antibodies:

• A B3 DNA-binding domain at the N-terminus that recognizes AuxRR elements

• An Auxin Response Factor domain in the middle region

• An AUX/IAA family domain at the C-terminus for protein-protein interactions

When developing antibodies, researchers should consider targeting unique epitopes outside highly conserved regions to prevent cross-reactivity with other ARF family members. The C-terminal region often provides more specificity for individual ARF proteins, while antibodies targeting the B3 domain might detect multiple ARF proteins.

What validation methods should be employed when using ARF18 antibodies?

When validating ARF18 antibodies, researchers should implement multiple approaches from the five validation pillars described for research antibodies:

• Orthogonal validation: Compare protein levels determined by antibody-dependent methods (Western blot) with antibody-independent methods (RT-qPCR or mass spectrometry)

• Genetic knockdown: Test antibody in wild-type versus ARF18-silenced or knockout plants (using VIGS, CRISPR/Cas9, or RNAi approaches)

• Recombinant expression: Use overexpression systems with tagged versions of ARF18 to confirm antibody specificity

• Independent antibodies: Compare results using different antibodies targeting distinct epitopes of ARF18

• Capture mass spectrometry: Perform immunoprecipitation followed by mass spectrometry to confirm binding specificity

It's essential to validate antibodies specifically for each application context (Western blot, immunoprecipitation, immunolocalization) as performance can vary between applications.

How should ARF18 antibodies be used in co-immunoprecipitation experiments to study protein interactions?

For co-immunoprecipitation (Co-IP) experiments studying ARF18 interactions:

• Sample preparation: Extract nuclear proteins from plant tissues (ARF18 is nuclear-localized)

• Cross-linking consideration: Use formaldehyde (1-1.5%) for in vivo cross-linking if studying transient interactions

• Antibody binding: Incubate nuclear extracts with ARF18 antibody coupled to protein A/G beads

• Validation controls: Include:

  • Input sample (pre-immunoprecipitation)

  • IgG control (non-specific antibody)

  • Extract from ARF18-silenced plants as negative control

• Elution and detection: Western blot using antibodies against potential interactors

This approach has been successfully used to demonstrate interaction between RhARF18 and RhHDA6 in roses, where tagged versions (RhARF18-MYC and RhHDA6-GFP) were co-expressed in Nicotiana benthamiana leaves, immunoprecipitated with anti-MYC antibody, and detected with anti-GFP antibody .

What techniques can be used to study ARF18 binding to DNA targets?

To study ARF18 binding to DNA:

• Electrophoretic Mobility Shift Assay (EMSA):

  • Express recombinant ARF18 (GST-tagged) in E. coli

  • Prepare biotin-labeled DNA probes containing AuxRR elements (consensus sequence GGTCCAT)

  • Include competition controls with unlabeled and mutated probes

  • Detect DNA-protein complexes by gel shift

• Chromatin Immunoprecipitation (ChIP):

  • Cross-link protein-DNA complexes in plant tissue

  • Immunoprecipitate with ARF18 antibody

  • Purify and analyze precipitated DNA by qPCR or sequencing

  • Measure histone modification status (H3K9/K14 acetylation) at binding sites

• DNA-Protein Interaction ELISA:

  • Immobilize double-stranded DNA containing AuxRR elements

  • Incubate with recombinant ARF18 or nuclear extracts

  • Detect binding with ARF18 antibody

RhARF18 was demonstrated to directly bind the AuxRR cis-element (GGTCCAT) in the RhAG promoter using EMSA, showing specific recognition that was competed by unlabeled but not mutated probes .

How does ARF18 expression change during plant development and stress responses?

ARF18 shows dynamic expression patterns:

• Developmental regulation:

  • In roses, RhARF18 expression decreases from early to late floral development stages

  • Expression is tissue-specific, with GUS reporter analysis showing expression in various tissues (for OsARF18)

• Stress responses:

  • OsARF18 is slightly induced by various abiotic stresses including salt, drought (PEG), oxidative stress (H₂O₂), ABA treatment, cadmium stress, and cold conditions

  • This suggests ARF18 involvement in stress response pathways

When studying ARF18 expression, researchers should consider both tissue specificity and developmental timing, using methods like RT-qPCR, in situ hybridization, or reporter gene fusions (e.g., promoter:GUS constructs).

What controls should be included when using ARF18 antibodies for immunolocalization studies?

For immunolocalization of ARF18:

• Essential controls:

  • Negative control: Primary antibody omission

  • Negative control: Tissues from ARF18-silenced or knockout plants

  • Positive control: Tissues with confirmed ARF18 overexpression

  • Peptide competition: Pre-incubate antibody with immunizing peptide

• Subcellular localization verification:

  • Co-staining with nuclear markers (DAPI)

  • Comparison with fluorescent protein fusions (e.g., ARF18-GFP)

• Fixation and permeabilization optimization:

  • Test multiple fixatives (paraformaldehyde, glutaraldehyde)

  • Optimize permeabilization for nuclear proteins (Triton X-100 concentration)

RhARF18 has been confirmed to localize to the nucleus through fluorescence co-localization studies, consistent with its function as a transcriptional regulator .

How can virus-induced gene silencing (VIGS) be used to study ARF18 function?

VIGS represents an effective approach for functional analysis of ARF18:

• VIGS vector design:

  • Select target regions specific to ARF18 (200-300bp)

  • Avoid conserved domains shared with other ARF proteins

  • Clone into appropriate VIGS vector (e.g., TRV-based)

• Experimental design:

  • Include empty vector control (e.g., TRV control)

  • Monitor silencing efficiency by RT-qPCR

  • Assess phenotypes related to known ARF18 functions:

    • Floral development (petal/stamen number in roses)

    • Herbicide resistance (in rice)

    • Seed weight and silique development (in rapeseed)

• Downstream analysis:

  • Expression analysis of target genes (e.g., RhAG in roses)

  • In situ hybridization to assess spatial expression changes

  • Quantitative phenotyping (e.g., counting floral organs)

In roses, RhARF18 silencing led to decreased petal number and increased stamen number, similar to the phenotype observed in RhPILS1-silenced plants, with concurrent upregulation of RhAG expression .

What methods can detect interactions between ARF18 and histone modification machinery?

To study ARF18 interactions with histone modifiers:

• Protein-protein interaction methods:

  • Yeast two-hybrid (Y2H) screening: Used successfully to identify RhHDA6 as an RhARF18 interactor

  • BiFC (Bimolecular Fluorescence Complementation): Demonstrated RhARF18-RhHDA6 interaction in plant cell nuclei

  • Co-IP coupled with Western blot: Confirmed interaction between RhARF18-MYC and RhHDA6-GFP in planta

• Functional verification:

  • ChIP assays to measure histone modification changes at ARF18 binding sites

  • ChIP-reChIP to demonstrate co-occupancy of ARF18 and histone modifiers at target loci

  • Dual-luciferase reporter assays with wild-type and mutant ARF18 proteins

Research in roses showed that RhARF18 recruits RhHDA6 to the RhAG promoter, leading to histone deacetylation (decreased H3K9/K14 acetylation) and transcriptional repression .

How can ARF18 structure-function relationships be studied using site-directed mutagenesis?

Advanced structure-function analysis of ARF18:

• Domain-specific mutations:

  • B3 DNA-binding domain: Mutations affecting AuxRR element recognition

  • Middle region: Modifications to transcriptional repression function

  • C-terminal domain: Alterations affecting protein-protein interactions (e.g., with HDA6)

• Functional assays:

  • DNA binding: EMSA with wild-type and mutant proteins

  • Transcriptional activity: Dual-luciferase reporter assays

  • Protein interactions: Y2H or BiFC with interaction partners

• In planta validation:

  • Complementation of ARF18-silenced or knockout plants with mutant variants

  • Phenotypic rescue assessment

For example, the 55-amino acid deletion identified in rapeseed ARF18 prevented homodimer formation and resulted in loss of binding activity, affecting seed weight and silique length .

What are the technical considerations when studying ARF18 in different plant species due to sequence variation?

When studying ARF18 across species, researchers should consider:

• Antibody cross-reactivity analysis:

  • Perform sequence alignment of ARF18 across target species

  • Design species-specific antibodies or select antibodies targeting conserved regions

  • Validate antibodies separately for each species

• Species-specific optimizations:

  • Extraction buffers: Adjust for different tissue types and secondary metabolites

  • Immunoprecipitation conditions: Optimize salt and detergent concentrations

  • Western blot: Adjust blocking conditions to minimize background

• Functional conservation assessment:

  • Compare phenotypic effects of ARF18 silencing/mutation across species

  • Analyze binding site preferences in different species

  • Test interspecies complementation

The provided search results show ARF18 functions in roses (RhARF18), rice (OsARF18), and rapeseed, with both conserved roles in auxin signaling and species-specific functions .

How can ChIP-seq be optimized for genome-wide identification of ARF18 binding sites?

For optimal ChIP-seq analysis of ARF18:

• Experimental design considerations:

  • Select appropriate tissues and developmental stages where ARF18 is active

  • Include input controls and IgG controls

  • Consider biological replicates to identify consistent binding sites

• ChIP optimization:

  • Crosslinking: Optimize time and formaldehyde concentration

  • Sonication: Adjust conditions to achieve 200-500bp DNA fragments

  • Antibody selection: Use ChIP-grade ARF18 antibodies validated for specificity

  • Enrichment assessment: Validate ChIP efficiency by qPCR of known targets before sequencing

• Data analysis pipeline:

  • Peak calling algorithms suitable for transcription factor binding

  • Motif discovery to identify ARF18 binding sequences

  • Integration with transcriptome data to correlate binding with gene expression changes

  • Comparison with histone modification data to assess chromatin state at binding sites

This approach would extend findings from targeted studies, such as the identified binding of RhARF18 to the AuxRR cis-element in the RhAG promoter in roses .

What are common pitfalls when using ARF18 antibodies and how can they be addressed?

Common challenges with ARF18 antibodies include:

• Cross-reactivity with other ARF proteins:

  • Solution: Use peptide competition assays

  • Test in ARF18 knockout/knockdown tissues

  • Compare against recombinant ARF18 protein standards

• Nuclear protein extraction difficulties:

  • Solution: Use specialized nuclear extraction buffers

  • Enhance nuclear membrane disruption (sonication/detergents)

  • Verify extraction efficiency with nuclear markers

• Weak signal in immunoblotting:

  • Solution: Optimize protein extraction with protease inhibitors

  • Increase protein loading for low-abundance tissues

  • Enhance detection with signal amplification systems

• Inconsistent immunoprecipitation results:

  • Solution: Pre-clear lysates thoroughly

  • Optimize antibody:bead ratios

  • Consider crosslinking antibodies to beads

Each antibody requires application-specific validation, as performance may vary between Western blotting, immunoprecipitation, and immunohistochemistry applications .

How can conflicting results between antibody-based and transcriptome-based ARF18 expression data be reconciled?

When facing discrepancies between protein and transcript data:

• Potential causes:

  • Post-transcriptional regulation (miRNAs targeting ARF18)

  • Post-translational modifications affecting antibody recognition

  • Protein stability differences across tissues/conditions

  • Antibody specificity issues

• Resolution strategies:

  • Temporal analysis: Assess time-course to detect delays between transcript and protein changes

  • Multiple antibodies: Use antibodies targeting different ARF18 epitopes

  • Orthogonal validation: Compare with tagged ARF18 versions detected with tag antibodies

  • Absolute quantification: Use recombinant protein standards for Western blot calibration

• Confirmatory approaches:

  • Mass spectrometry-based protein quantification

  • Polysome profiling to assess translation efficiency

  • Protein half-life measurements using cycloheximide chase

For example, in rice, OsARF18 expression showed responsiveness to abiotic stresses at the transcript level , which should be confirmed at the protein level using validated antibodies.

How can ARF18 antibodies be applied to study protein complex dynamics during auxin signaling?

Advanced applications for studying ARF18 complexes:

• Proximity-dependent labeling approaches:

  • BioID or TurboID fusion with ARF18 to identify proximal proteins

  • APEX2-ARF18 fusions for temporal mapping of interaction networks

  • Quantitative analysis of complex composition changes after auxin treatment

• Single-molecule imaging:

  • Antibody-based single-molecule tracking in living cells

  • Super-resolution microscopy to visualize ARF18 complex formation

  • FRET-based approaches to measure interaction dynamics

• Chromatin interaction studies:

  • ChIP-seq combined with protein complex analysis

  • HiChIP or PLAC-seq to connect ARF18 binding with 3D chromatin structure

  • Sequential ChIP to identify co-occupancy with other factors

This builds on findings that ARF18 interacts with histone modifiers like HDA6 and affects transcription of target genes like RhAG in roses and glutamine synthetase genes in rice .

What potential applications exist for ARF18 antibodies in agricultural biotechnology research?

ARF18 antibodies in agricultural research applications:

• Trait development monitoring:

  • Tracking ARF18 protein levels in crop varieties with enhanced yield traits

  • Monitoring ARF18-regulated pathways during seed development in crops

  • Studying ARF18 status during herbicide response in resistant varieties

• Stress response characterization:

  • Analyzing ARF18 protein dynamics during drought, salt, or pathogen stress

  • Correlating ARF18 activity with stress adaptation mechanisms

  • Comparing ARF18 status between stress-tolerant and susceptible varieties

• Developmental phenotyping:

  • High-throughput screening of ARF18 levels in breeding populations

  • Associating ARF18 protein abundance with desired agricultural traits

  • Early detection of phenotypic outcomes in modified lines

These applications are supported by findings linking ARF18 to agriculturally important traits like seed weight in rapeseed and herbicide resistance in rice .