ARF15 Antibody

What is ARF15 and what cellular functions does it regulate?

ARF15 (Auxin response factor 15) is a plant transcription factor that specifically binds to the DNA sequence 5'-TGTCTC-3' found in auxin-responsive promoter elements (AuxREs). It functions as a critical regulator in the auxin signaling pathway, which controls numerous aspects of plant growth and development. ARF15 is also known by several alternative names including ETTIN-like protein 1, OsETTIN1, Os05g0563400, and LOC_Os05g48870. This protein is primarily studied in plant models such as rice (Oryza sativa), Arabidopsis thaliana, and maize (Zea mays), where it contributes to auxin-mediated developmental processes . Unlike the similarly named ARF1 (ADP-ribosylation factor 1), which is a small GTPase involved in vesicular trafficking in mammals, ARF15 functions specifically within plant systems as a transcriptional regulator that mediates auxin responses essential for normal plant development.

How do ARF15 antibodies differ from other plant transcription factor antibodies?

ARF15 antibodies are specifically designed to recognize epitopes unique to the Auxin response factor 15 protein, distinguishing it from other members of the ARF family and other plant transcription factors. Unlike antibodies against constitutively expressed proteins, ARF15 antibodies target a protein whose expression may vary considerably depending on developmental stage, tissue type, and environmental stimuli. Currently available commercial ARF15 antibodies include mouse monoclonal antibodies with reactivity toward rice, Arabidopsis thaliana, and maize variants of the protein . These antibodies differ from antibodies against other plant transcription factors in their epitope specificity, cross-reactivity profiles with different plant species, and optimal application conditions. Researchers should note that validation of ARF15 antibodies typically requires careful controls including wild-type versus ARF15 knockout plant tissues to confirm specificity, particularly when studying closely related ARF family members.

What are the optimal applications for ARF15 antibodies in plant molecular biology research?

ARF15 antibodies are valuable tools for multiple experimental applications in plant molecular biology. Based on current research protocols, the most reliable applications include:

• Western Blot (WB): For detecting and quantifying ARF15 protein expression levels in plant tissue extracts. Typically run under reducing conditions using 10-12% SDS-PAGE gels.

• ELISA: For quantitative measurement of ARF15 in plant extracts, offering high sensitivity for detecting low abundance proteins.

• Immunofluorescence (IF): For visualizing cellular and subcellular localization of ARF15, particularly during developmental transitions .

For optimal results in any application, researchers should consider:

• Tissue-specific expression patterns of ARF15 across different plant organs

• Developmental timing, as ARF15 expression fluctuates during plant development

• Potential post-translational modifications that may affect antibody recognition

• Cross-reactivity with other ARF family members, necessitating validation with appropriate controls

Advanced applications may include chromatin immunoprecipitation (ChIP) assays to identify ARF15 binding sites on DNA, though this requires highly specific antibodies and careful optimization of experimental conditions.

What extraction and sample preparation protocols maximize ARF15 antibody performance?

Optimizing protein extraction and sample preparation is critical for successful ARF15 antibody applications. The following protocol has been demonstrated to yield reliable results:

• Tissue harvest and preservation:

  • Collect fresh plant tissue (preferably young, actively growing tissue where auxin signaling is active)

  • Flash freeze immediately in liquid nitrogen

  • Store at -80°C until processing

• Protein extraction buffer optimization:

  • Use a buffer containing: 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1% Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS

  • Add fresh protease inhibitors (e.g., 1 mM PMSF, 1 μg/ml leupeptin, 1 μg/ml aprotinin)

  • Include phosphatase inhibitors if studying phosphorylated forms

  • Add 5 mM DTT to maintain reducing conditions

• Extraction procedure:

  • Grind tissue thoroughly in liquid nitrogen using a mortar and pestle

  • Add 3-5 volumes of extraction buffer per gram of tissue

  • Homogenize and incubate with gentle agitation for 30 minutes at 4°C

  • Centrifuge at 15,000 × g for 15 minutes at 4°C

  • Collect supernatant and determine protein concentration

• Sample preparation for immunoblotting:

  • Dilute samples to equal protein concentration

  • Mix with 4× Laemmli buffer containing β-mercaptoethanol

  • Heat at 95°C for 5 minutes

  • Load 20-50 μg total protein per lane

This protocol minimizes protein degradation and maximizes extraction efficiency while preserving the native epitopes recognized by ARF15 antibodies.

How can researchers utilize ARF15 antibodies to investigate auxin-mediated transcriptional regulation?

Investigating auxin-mediated transcriptional regulation using ARF15 antibodies requires sophisticated experimental approaches that can capture the dynamic nature of ARF15 activity. Advanced researchers can implement the following strategies:

• Chromatin Immunoprecipitation (ChIP) followed by sequencing (ChIP-seq):

  • Cross-link proteins to DNA in plant tissues at different developmental stages or after auxin treatment

  • Sonicate chromatin to 200-500 bp fragments

  • Immunoprecipitate using ARF15 antibodies with validated specificity

  • Sequence precipitated DNA to identify genome-wide ARF15 binding sites

  • Analyze enrichment of auxin-responsive elements (AuxREs)

• Co-immunoprecipitation (Co-IP) to identify protein interaction partners:

  • Prepare native protein extracts under non-denaturing conditions

  • Immunoprecipitate using ARF15 antibodies

  • Identify co-precipitated proteins through mass spectrometry

  • Validate interactions through reciprocal Co-IPs and in vitro binding assays

• Time-course immunoblotting after auxin treatment:

  • Treat plant tissues with synthetic auxins (e.g., 2,4-D, NAA) or natural auxin (IAA)

  • Harvest tissues at multiple time points (0, 15, 30, 60, 120 minutes)

  • Perform quantitative Western blot analysis with ARF15 antibodies

  • Correlate ARF15 protein levels or modifications with auxin-responsive gene expression

• Proximity-dependent biotin identification (BioID) or TurboID:

  • Generate plants expressing ARF15 fused to a biotin ligase

  • Identify proteins that interact with ARF15 in vivo through biotinylation

  • Purify biotinylated proteins using streptavidin beads

  • Identify interaction partners by mass spectrometry

  • Validate findings using ARF15 antibodies in orthogonal approaches

These approaches enable researchers to construct comprehensive models of ARF15's role in auxin signaling networks and transcriptional regulation of plant development.

What controls are essential when validating ARF15 antibody specificity in plant research?

Rigorous validation of ARF15 antibody specificity is critical for generating reliable research data. Essential controls include:

• Genetic controls:

  • Wild-type versus arf15 knockout/knockdown plants

  • Complementation lines expressing tagged ARF15 (e.g., ARF15-GFP)

  • Overexpression lines with elevated ARF15 levels

• Peptide competition assays:

  • Pre-incubate ARF15 antibody with excess immunizing peptide

  • Compare immunoblot or immunostaining patterns with and without peptide blocking

  • Specific signals should be significantly reduced or eliminated

• Cross-reactivity assessment:

  • Test antibody against recombinant proteins from related ARF family members

  • Perform immunoblots on tissues from plants expressing different ARF proteins

  • Create a cross-reactivity table documenting specificity across ARF family

• Application-specific controls:

  • For IF: Include secondary antibody-only controls and non-relevant primary antibody controls

  • For WB: Include molecular weight markers and positive control samples

  • For IP: Include IgG isotype controls and beads-only controls

• Heterologous expression validation:

  • Express ARF15 in heterologous systems (e.g., bacteria, yeast)

  • Confirm antibody detection of purified recombinant protein

  • Compare detection limits and signal-to-noise ratios

How do post-translational modifications affect ARF15 antibody recognition and experimental outcomes?

Post-translational modifications (PTMs) can significantly impact ARF15 antibody recognition, leading to potential misinterpretation of experimental results. Researchers should consider:

• Common PTMs affecting ARF15 recognition:

  • Phosphorylation: May alter epitope accessibility or antibody binding affinity

  • Ubiquitination: Can mask epitopes or alter protein migration patterns

  • SUMOylation: May change protein conformation affecting antibody access

• Strategies to address PTM interference:

  • Use phosphatase treatment of samples to remove phosphorylation

  • Compare native versus denatured protein detection efficiency

  • Generate modification-specific antibodies for studying specific PTM states

  • Use multiple antibodies targeting different epitopes to confirm results

• Experimental design considerations:

  • Include both reducing and non-reducing conditions in parallel

  • Compare young versus mature tissues where PTM profiles may differ

  • Design time-course experiments after hormone treatments that may induce PTMs

  • Consider differential extraction methods to preserve or remove specific modifications

• Interpretation guidelines:

  • Multiple bands may represent different modification states rather than non-specific binding

  • Absence of signal may indicate epitope masking rather than absence of protein

  • Changes in apparent molecular weight may reflect modifications rather than protein isoforms

What are the common technical challenges when using ARF15 antibodies and how can they be resolved?

Researchers frequently encounter several challenges when working with ARF15 antibodies. Here are evidence-based solutions:

For particularly challenging applications, researchers can implement these advanced approaches:

• Use tandem antibody approaches targeting different epitopes of ARF15

• Implement stepwise optimization of buffer conditions using design of experiments (DOE) methodology

• Consider alternate fixation methods for difficult tissues (e.g., ethanol-acetic acid fixation instead of formaldehyde)

• Validate findings with orthogonal techniques not dependent on antibody recognition

How should researchers interpret conflicting data from different ARF15 antibody applications?

When faced with conflicting results across different applications using ARF15 antibodies, researchers should implement a systematic analytical approach:

• Categorize discrepancies by type:

  • Detection versus non-detection in different applications

  • Different molecular weights observed across techniques

  • Conflicting subcellular localization patterns

  • Inconsistent protein abundance measurements

• Technical validation hierarchy:

  • Establish which technique provides the most reliable data for your specific question

  • Consider the resolution limitations of each method (e.g., IF provides spatial information but poorer quantification than WB)

  • Assess whether discrepancies arise from technical artifacts or genuine biological phenomena

• Resolution strategies:

  • Genetic complementation: Compare results with tagged ARF15 expressed in arf15 mutant background

  • Antibody validation: Test multiple antibodies recognizing different epitopes

  • Application optimization: Refine protocols for each technique to ensure optimal conditions

  • Orthogonal validation: Use non-antibody methods (e.g., RNA-seq, mass spectrometry) to corroborate findings

• Data integration framework:

  • Develop a molecular model that accounts for observed discrepancies

  • Consider biological context (tissue type, developmental stage, environmental conditions)

  • Evaluate whether conflicting data reflects dynamic biological processes rather than technical issues

  • Document conditions under which different results are obtained to identify pattern-revealing variables

How do monoclonal versus polyclonal ARF15 antibodies compare in plant research applications?

The choice between monoclonal and polyclonal ARF15 antibodies significantly impacts experimental outcomes in plant research:

Currently available mouse monoclonal antibodies against ARF15 have demonstrated reliable performance in Western blot and ELISA applications . These antibodies show reactivity across multiple plant species including rice, Arabidopsis thaliana, and maize. For applications requiring higher sensitivity or where epitope masking might occur, polyclonal antibodies may offer advantages, though they require more extensive validation to ensure specificity against other ARF family members.

When selecting between monoclonal and polyclonal options, researchers should prioritize antibodies that have been validated specifically for their plant species and application of interest, ideally with published validation data.

What are the critical factors to consider when selecting an ARF15 antibody for specific plant species?

Selecting the appropriate ARF15 antibody for different plant species requires consideration of several critical factors:

• Sequence homology analysis:

  • Compare ARF15 amino acid sequences across target plant species

  • Identify conserved versus variable regions that might affect epitope recognition

  • Select antibodies raised against epitopes with high conservation for cross-species applications

• Species validation documentation:

  • Review published literature and technical documentation for species-specific validation

  • Current commercial ARF15 antibodies have validated reactivity for rice, Arabidopsis, and maize

  • Request validation data from manufacturers for non-model plant species

• Application compatibility matrix:

  • Determine if the antibody has been validated for your specific application in your species

  • For example, an antibody may work for Western blot in Arabidopsis but not for immunofluorescence in rice

• Tissue-specific considerations:

  • Assess antibody performance in different tissue types (root, shoot, flowers, etc.)

  • Consider tissue-specific protein expression levels and extraction challenges

  • Account for tissue-specific background or cross-reactivity issues

• Fixation and extraction compatibility:

  • Different plant species may require species-specific fixation protocols

  • Cell wall composition varies across species, affecting extraction efficiency

  • Secondary metabolites in certain species may interfere with antibody binding

For researchers working with non-model plants, preliminary validation experiments are essential, including:

• Side-by-side comparison with model plant positive controls

• Peptide competition assays to confirm specificity

• Correlation of protein detection with transcript expression data

• Use of heterologous expression systems to verify antibody performance

How can ARF15 antibodies be utilized in emerging plant biotechnology applications?

ARF15 antibodies are becoming increasingly valuable tools in cutting-edge plant biotechnology applications:

• CRISPR-engineered ARF15 variants:

  • Antibodies can verify expression of edited ARF15 proteins

  • Enable phenotype-to-protein correlation in plants with precise ARF15 mutations

  • Validate knock-in of tagged or modified ARF15 variants

• Single-cell plant proteomics:

  • ARF15 antibodies conjugated to barcoded tags for single-cell protein analysis

  • Reveal cell-type specific ARF15 expression patterns in complex tissues

  • Track auxin response heterogeneity at the single-cell level during development

• Synthetic auxin signaling circuits:

  • Monitor ARF15 protein dynamics in synthetic biology applications

  • Validate orthogonal auxin signaling components in engineered plants

  • Quantify protein-level feedback in synthetic hormone response networks

• Environmental stress adaptation studies:

  • Track ARF15 protein modifications under various abiotic stresses

  • Correlate ARF15 activity with stress adaptation phenotypes

  • Identify stress-specific protein interaction networks using ARF15 antibodies

• Multiplexed protein detection platforms:

  • Include ARF15 antibodies in antibody panels for simultaneous detection of multiple auxin signaling components

  • Implement in high-throughput phenotyping platforms to correlate protein expression with growth parameters

  • Combine with other hormone pathway antibodies to study signaling crosstalk

These emerging applications represent the frontier of plant molecular biology research and demonstrate the continued importance of well-validated ARF15 antibodies in advancing our understanding of plant development and agricultural improvement.

What methodological advances are enhancing the utility of ARF15 antibodies in plant developmental biology?

Recent methodological innovations are significantly expanding the applications of ARF15 antibodies in plant developmental biology:

• Clearing-compatible immunostaining protocols:

  • New fixation and permeabilization methods preserve ARF15 epitopes while allowing tissue clearing

  • Enable whole-mount 3D visualization of ARF15 distribution throughout intact plant organs

  • Reveal spatial relationships between ARF15 localization and developmental patterning

• Super-resolution microscopy applications:

  • Specially conjugated ARF15 antibodies compatible with STORM, PALM, and STED microscopy

  • Resolve subnuclear localization patterns of ARF15 at nanometer resolution

  • Track ARF15 clustering at specific chromatin regions during transcriptional activation

• Mass cytometry (CyTOF) adaptation:

  • Metal-conjugated ARF15 antibodies for high-dimensional protein analysis

  • Simultaneous quantification of multiple transcription factors including ARF15

  • Single-cell resolution of auxin response network components

• Microfluidic immunoassays:

  • Miniaturized systems requiring minimal sample amounts (critical for rare cell types)

  • Rapid analysis of ARF15 in multiple samples or conditions

  • Integration with live-imaging systems for correlative protein-phenotype analysis

• Proximity labeling advances:

  • ARF15 antibodies for validation of TurboID or APEX2 proximity labeling results

  • Confirmation of in vivo protein-protein interactions identified through proximity labeling

  • Spatial verification of interaction networks in specific cellular compartments

These methodological advances are transforming our ability to study ARF15 dynamics in plant development, enabling unprecedented insights into the spatiotemporal regulation of auxin response and its role in controlling plant growth and morphogenesis.