ARF19 Antibody

What is ARF19 and why is it important in plant research?

ARF19 is a class-A Auxin Response Factor transcription factor that functions as a transcriptional activator in auxin signaling pathways. It works coordinately with the closely-related ARF7 to play essential roles in auxin-mediated plant development, particularly in lateral root emergence and development . ARF19 is critical for plant responses to the phytohormone auxin, making it an important target for researchers studying plant development, stress responses, and hormone signaling networks.

Antibodies against ARF19 are invaluable tools for researchers seeking to understand the spatiotemporal dynamics of this protein, its post-translational modifications, protein-protein interactions, and regulatory mechanisms controlling its abundance and activity. Recent research has shown that ARF19 protein accumulation is developmentally regulated and dependent on the 26S-proteasome, highlighting the importance of studying its protein dynamics .

What sample preparation techniques work best for ARF19 antibody applications?

For optimal ARF19 antibody applications, sample preparation should account for the nuclear localization of this transcription factor and its tendency to form protein condensates. For immunoblotting:

• Harvest fresh plant tissue (preferably root tissue for lateral root studies) and flash-freeze in liquid nitrogen.

• Grind tissue to a fine powder while maintaining freezing temperatures.

• Extract proteins using a nuclear extraction buffer containing:

  • 50 mM Tris-HCl (pH 7.5)

  • 150 mM NaCl

  • 0.5% Triton X-100

  • 1 mM EDTA

  • 1 mM DTT

  • Protease inhibitor cocktail

  • 10 μM MG132 (to prevent degradation during extraction)

For immunolocalization studies, fix tissue samples in 4% paraformaldehyde for 1 hour at room temperature, followed by careful washing and permeabilization steps. When studying ARF19 condensates, minimal fixation times are recommended to preserve the native distribution of protein condensates .

How can researchers validate ARF19 antibody specificity?

Validating ARF19 antibody specificity is crucial, particularly due to the sequence similarity between ARF19 and other ARF family members. Use these approaches for comprehensive validation:

• Western blot analysis using knockout mutants: Compare wild-type plants with arf19 mutants (such as arf19-1). A specific antibody should show absence of signal in the knockout line .

• Peptide competition assay: Pre-incubate the antibody with the immunizing peptide before applying to samples. This should eliminate specific signal.

• Recombinant protein controls: Use purified recombinant ARF19 protein as a positive control and other recombinant ARF proteins to assess cross-reactivity.

• Correlation with fluorescent fusion proteins: In transgenic lines expressing YFP-ARF19 or similar fusion proteins, confirm that antibody signal correlates with fluorescent protein localization .

• Cross-species validation: If the antibody is claimed to work across multiple plant species, test specificity in each species separately.

What are common pitfalls when working with ARF19 antibodies?

Several challenges may arise when working with ARF19 antibodies:

• Rapid protein degradation: ARF19 protein is subject to proteasomal degradation. Include proteasome inhibitors (e.g., MG132) in extraction buffers and process samples quickly .

• Cross-reactivity with ARF7: Due to high sequence similarity, antibodies may cross-react with ARF7. Always validate using appropriate controls, including arf7 and arf19 single mutants, and the arf7 arf19 double mutant.

• Developmental regulation: ARF19 protein levels change during development, especially during lateral root formation. Ensure consistent developmental staging when comparing samples .

• Post-translational modifications: Modifications can affect antibody binding. Consider using phospho-specific antibodies if studying ARF19 phosphorylation states.

• Protein condensation: ARF19 forms protein condensates that may make epitopes inaccessible. Optimize fixation and extraction protocols to preserve and/or solubilize these structures as needed for your experimental goals .

How can researchers effectively study ARF19 protein degradation dynamics?

ARF19 protein degradation is regulated by the 26S-proteasome, and recent research has identified AFF1 (AUXIN RESPONSE FACTOR F-BOX1) as a key regulator of this process . To study degradation dynamics:

• Cycloheximide chase assays: Treat plants with cycloheximide to inhibit protein synthesis, then collect samples at various time points to track ARF19 degradation rates. Compare wild-type plants with mutants in the degradation pathway (e.g., aff1 mutants).

• Proteasome inhibitor treatments: Apply MG132 to block proteasomal degradation and monitor ARF19 accumulation over time using immunoblotting .

• Ubiquitination assays: Use immunoprecipitation with ARF19 antibodies followed by ubiquitin-specific antibody detection to assess ubiquitination status.

• Fluorescence recovery after photobleaching (FRAP): In plants expressing fluorescent-tagged ARF19, use FRAP to measure protein turnover rates in different cellular compartments or developmental contexts.

• Pulse-chase experiments: Employ inducible expression systems coupled with time-course sampling to measure half-life in different genetic backgrounds or treatment conditions.

When conducting these experiments, researchers should consider the F-box protein AFF1's role, as it physically interacts with ARF19 and promotes its degradation. Mutants defective in AFF1 hyperaccumulate ARF19 protein and display attenuated auxin responsiveness .

What approaches are recommended for studying ARF19 protein condensates?

Recent research has revealed that ARF19 can form protein condensates, and increased condensation correlates with attenuated auxin responses . To effectively study these condensates:

• Live cell imaging: Use plants expressing YFP-ARF19 or other fluorescent fusions to visualize condensate formation in real-time. Time-lapse imaging can capture condensate dynamics.

• Immunofluorescence with minimal fixation: Employ mild fixation protocols to preserve condensate structure while enabling antibody detection.

• Co-localization studies: Use antibodies or fluorescent reporters for known condensate markers alongside ARF19 detection to characterize the nature of these structures.

• Biochemical fractionation: Develop protocols to isolate and characterize ARF19-containing condensates, followed by proteomic analysis.

• Quantitative image analysis: Implement computational approaches to quantify condensate number, size, and intensity across different conditions and genotypes .

Researchers should be aware that in aff1 mutants, which hyperaccumulate ARF19 protein, there is an increased number of ARF19 condensates compared to wild type. This increased condensation correlates with attenuated auxin responses, suggesting that proper regulation of ARF19 protein levels is crucial for maintaining appropriate auxin responsiveness .

How can researchers distinguish between ARF19 and ARF7 in experimental systems?

Distinguishing between ARF19 and ARF7 is challenging due to their high sequence similarity and overlapping functions in auxin-mediated development. Consider these approaches:

• Epitope selection: Design antibodies against unique regions of ARF19 that differ from ARF7. The C-terminal domain often shows greater divergence between ARF family members.

• Genetic backgrounds: Utilize arf7 single mutants when studying ARF19, and vice versa, to eliminate potential cross-reactivity.

• Isoform-specific RT-qPCR: Complement protein-level studies with transcript-level analysis using highly specific primers.

• Immunoprecipitation followed by mass spectrometry: This approach can definitively identify which ARF protein has been captured by the antibody.

• Differential expression patterns: Although ARF7 and ARF19 have overlapping functions, they can show distinct expression patterns in certain tissues or developmental stages. Use this knowledge for contextual validation .

When conducting experiments requiring isoform specificity, consider creating an epitope-tagged version of ARF19 in the arf19 mutant background, allowing the use of commercial tag-specific antibodies with validated specificity.

What methodological considerations are important when studying ARF19's role in lateral root development?

Lateral root development involves complex spatiotemporal regulation of ARF19, with important interactions with other factors like LBD29 . When studying ARF19's role:

• Developmental staging: Carefully stage lateral root primordia using established classifications (Stages I-VIII) to ensure consistent comparisons.

• Cell-type specific analysis: ARF19 functions in multiple cell types during lateral root emergence. Use cell-type specific promoters driving fluorescent ARF19 fusions for spatial resolution.

• Temporal resolution: Consider inducible systems to manipulate ARF19 levels at specific developmental stages.

• Co-visualization with interaction partners: Employ dual immunolocalization or fluorescent protein fusions to study ARF19 alongside partners like LBD29, which is expressed in lateral root primordia and cells directly overlying new primordia .

• Quantitative phenotyping: Establish robust methodologies for quantifying lateral root emergence defects, including:

  • Emerged lateral root density

  • Developmental stage distribution of primordia

  • Emergence rate over time

  • Cell wall modifications in overlying tissues

Research shows that LBD29 functions as a direct positive regulator of LAX3 auxin-dependent expression downstream of ARF7. LAX3 is an auxin influx carrier that facilitates lateral root emergence by promoting cell wall remodeling in overlying tissues .

How can researchers optimize western blot protocols for ARF19 detection?

Optimizing western blot protocols for ARF19 detection requires addressing several specific challenges:

• Protein extraction buffer optimization:

  • Use HEPES or Tris buffer (pH 7.5-8.0)

  • Include 0.5-1% NP-40 or Triton X-100

  • Add 150-300 mM NaCl

  • Include protease inhibitor cocktail

  • Add 10-50 μM MG132 to prevent degradation

  • Include 1-5 mM DTT or β-mercaptoethanol as reducing agents

• Gel percentage selection: Use 8-10% acrylamide gels for optimal resolution of ARF19 (approximately 80-90 kDa).

• Transfer conditions: Use semi-dry transfer at 15V for 1 hour or wet transfer at 30V overnight at 4°C for efficient transfer of larger proteins.

• Blocking optimization: Test both 5% BSA and 5% non-fat dry milk in TBST to determine optimal blocking conditions that maximize signal-to-noise ratio.

• Antibody dilution and incubation: Start with 1:1000 dilution for primary antibody, incubating overnight at 4°C. For secondary antibody, use 1:5000-1:10000, incubating for 1 hour at room temperature.

If signal is weak, consider using enhanced chemiluminescence (ECL) detection systems with extended exposure times, or switch to fluorescent secondary antibodies for increased sensitivity and quantitative analysis.

What strategies are effective for co-immunoprecipitation of ARF19 with interaction partners?

Co-immunoprecipitation (Co-IP) of ARF19 with interaction partners requires specific considerations:

• Buffer composition: Use a gentle lysis buffer to preserve protein-protein interactions:

  • 50 mM Tris-HCl (pH 7.5)

  • 150 mM NaCl

  • 0.5% NP-40 or 0.3% Triton X-100

  • 1 mM EDTA

  • 10% glycerol

  • Protease inhibitor cocktail

  • 10-50 μM MG132

  • Phosphatase inhibitors if studying phosphorylation-dependent interactions

• Cross-linking considerations: For transient or weak interactions, consider using formaldehyde (0.5-1%) or DSP (dithiobis(succinimidyl propionate)) cross-linking prior to extraction.

• Antibody selection: Use validated antibodies with high specificity. Consider using tag-specific antibodies if working with epitope-tagged ARF19 versions.

• Optimizing pull-down conditions:

  • Pre-clear lysates with protein A/G beads to reduce non-specific binding

  • Optimize antibody concentration and incubation time

  • Include appropriate controls (IgG control, input sample)

  • Consider sequential IPs for higher purity

• Verification by reciprocal Co-IP: Confirm interactions by performing the Co-IP in reverse, using antibodies against the interaction partner to pull down ARF19.

Research shows that ARF19 interacts with the F-box protein AFF1, which regulates its stability . This interaction could serve as a positive control for optimizing Co-IP protocols.

How should researchers address contradictory data in ARF19 localization studies?

When faced with contradictory data regarding ARF19 localization:

• Evaluate fixation artifacts: Different fixation methods can affect protein localization. Compare results from multiple fixation protocols and include live-cell imaging when possible.

• Consider developmental context: ARF19 localization may vary with tissue type, developmental stage, and environmental conditions. Ensure precise documentation of these factors.

• Assess antibody specificity in the specific context: Antibodies may show different specificity in different applications (western blot vs. immunofluorescence) or under different fixation conditions.

• Compare with fluorescent protein fusions: Validate antibody-based localization with fluorescent protein fusions, being mindful that tags may occasionally affect localization.

• Examine technical variability: Systematically evaluate:

  • Sample preparation method

  • Antibody lot variability

  • Detection system sensitivity

  • Image acquisition parameters

  • Analysis thresholds and settings

Recent research demonstrates that ARF19 forms protein condensates, which can appear as distinct nuclear foci . This condensation behavior might explain discrepancies in localization data, as different experimental conditions could affect condensate formation or preservation.

What statistical approaches are recommended for analyzing ARF19 protein level changes?

When analyzing ARF19 protein level changes across different conditions or genotypes:

• Normalization strategies:

  • Use consistent loading controls (HSC70 has been used successfully in ARF19 studies)

  • Consider multiple normalization references (total protein stain plus specific loading control)

  • For fluorescence-based detection, use ratiometric approaches when possible

• Appropriate statistical tests:

  • For comparing two conditions: Student's t-test for normally distributed data

  • For multiple comparisons: ANOVA followed by post-hoc tests (e.g., LSD multiple range tests as used in ARF19 research)

  • For non-normally distributed data: Non-parametric alternatives (Mann-Whitney, Kruskal-Wallis)

• Biological replicates: Include at least three independent biological replicates, with technical replication within each.

• Time-course analysis: For degradation studies or dynamic responses, consider:

  • Area under curve (AUC) analysis

  • Curve-fitting to determine half-life

  • Rate of change calculations at different time points

• Correlation analysis: When examining relationships between ARF19 levels and physiological responses, use appropriate correlation methods and regression analysis.

Studies have shown that quantification of western blot data can effectively demonstrate significant differences in ARF19 protein accumulation between wild-type and mutant plants (e.g., aff1 mutants) .

How can researchers effectively interpret changes in ARF19 protein condensation patterns?

Interpreting changes in ARF19 protein condensation patterns requires sophisticated analysis:

• Quantitative parameters to measure:

  • Number of condensates per nucleus

  • Size distribution of condensates

  • Fluorescence intensity within condensates

  • Spatial distribution of condensates within the nucleus

  • Dynamics of condensate formation and dissolution

• Correlation with functional outcomes:

  • Associate condensate patterns with transcriptional readouts of ARF19 activity

  • Examine relationship between condensation and auxin responsiveness

  • Compare condensation patterns across mutants with altered auxin sensitivity

• Advanced imaging analysis:

  • Implement machine learning approaches for unbiased condensate detection

  • Use tracking algorithms for time-lapse analysis

  • Apply 3D reconstruction for volumetric analysis

• Control comparisons:

  • Compare with other transcription factors known to form or not form condensates

  • Evaluate condensation under varying auxin concentrations

  • Assess impact of protein accumulation levels on condensation patterns

Research has shown that aff1 mutants, which hyperaccumulate ARF19 protein, display increased numbers of ARF19 condensates compared to wild type, and this correlates with attenuated auxin responses . This suggests that proper regulation of ARF19 protein levels is crucial for maintaining appropriate condensation patterns and auxin responsiveness.

What approaches help distinguish between direct and indirect effects when studying ARF19 regulatory networks?

Distinguishing direct from indirect effects in ARF19 regulatory networks requires multiple complementary approaches:

• Chromatin immunoprecipitation (ChIP) assays:

  • Use ARF19-specific antibodies for ChIP-qPCR or ChIP-seq

  • Identify direct binding sites in promoters of putative target genes

  • Include positive controls like known ARF19 targets

  • Compare binding sites with known auxin response elements (AuxREs)

• Rapid induction systems:

  • Use systems combining hormone-inducible nuclear translocation with transcriptional inhibitors

  • Distinguish primary from secondary response genes

  • Measure response kinetics to identify likely direct targets

• In vitro binding assays:

  • EMSA (electrophoretic mobility shift assay) to confirm direct DNA binding

  • DNA affinity purification to identify binding sequences

  • Surface plasmon resonance for binding kinetics

• Genetic approaches:

  • Compare single, double, and higher-order mutants to identify epistatic relationships

  • Use inducible artificial microRNA or CRISPR interference for temporal control

  • Create separator lines with mutations in binding sites of putative targets

Research on ARF7 and LBD29 demonstrates how indirect regulation can be identified: ARF7 was initially thought to directly regulate LAX3, but experimental evidence showed that ARF7 actually regulates LAX3 indirectly through LBD29, which functions as a direct positive regulator of LAX3 expression .

How can researchers design experiments to study cell-type specific functions of ARF19?

Studying cell-type specific functions of ARF19 requires sophisticated experimental approaches:

• Cell-type specific expression systems:

  • Use promoters active in specific cell types (e.g., cortex, endodermis, epidermis)

  • Drive ARF19 expression in arf19 mutant background for complementation studies

  • Combine with fluorescent markers for visualization

• Cell-type specific perturbation:

  • Implement cell-type specific CRISPR systems

  • Use artificial microRNAs driven by cell-type specific promoters

  • Apply methods for cell-type specific protein degradation (e.g., dTAG system)

• Single-cell approaches:

  • Perform single-cell RNA-seq on root tissues to identify cell-type specific transcriptional responses

  • Use protoplasting and FACS to isolate specific cell populations

  • Apply spatial transcriptomics for in situ gene expression analysis

• Cell-type specific protein-protein interaction studies:

  • Implement split fluorescent protein systems under cell-type specific promoters

  • Use proximity labeling approaches (BioID, TurboID) in specific cell types

• Microscopy-based analysis:

  • Perform high-resolution confocal imaging of fluorescent ARF19 reporters

  • Use light-sheet microscopy for 3D + time analysis in intact roots

  • Implement FRET sensors to study ARF19 interactions in specific cells

Research shows that LBD29, which acts downstream of ARF7 in regulating lateral root emergence, is expressed in lateral root primordia and cells directly overlying new primordia, while LAX3 is expressed in cortical cells overlying new primordia but not in endodermal cells . These specific expression patterns highlight the importance of cell-type resolution when studying ARF19 and related factors.

What approaches are effective for studying post-translational modifications of ARF19?

Studying post-translational modifications (PTMs) of ARF19 requires specialized techniques:

• Phosphorylation analysis:

  • Use phospho-specific antibodies if available

  • Perform immunoprecipitation followed by phospho-specific western blotting

  • Employ mass spectrometry for phosphosite mapping

  • Create phospho-mimetic and phospho-dead mutants to test functional consequences

• Ubiquitination studies:

  • Detect ubiquitinated ARF19 using ubiquitin-specific antibodies following immunoprecipitation

  • Use tagged ubiquitin to pull down ubiquitinated proteins and detect ARF19

  • Analyze ubiquitination patterns in aff1 mutants vs. wild type

  • Create lysine-to-arginine mutants at potential ubiquitination sites

• SUMOylation analysis:

  • Perform immunoprecipitation followed by SUMO-specific western blotting

  • Use SUMO-specific proteases to confirm modification

  • Identify SUMO interaction motifs and create mutant versions

• PTM crosstalk:

  • Investigate how different modifications influence each other

  • Study temporal dynamics of modifications in response to auxin

  • Examine modification patterns in different developmental contexts

• Proteomic approaches:

  • Use immunoprecipitation coupled with mass spectrometry for comprehensive PTM mapping

  • Apply targeted proteomics (PRM/MRM) for quantitative analysis of specific modifications

  • Implement stable isotope labeling for comparative PTM analysis

Research has shown that ARF19 protein accumulation is regulated by the F-box protein AFF1, suggesting ubiquitin-mediated proteasomal degradation as a key regulatory mechanism . Understanding the sites and dynamics of ubiquitination would provide deeper insights into this process.