ARR5 Antibody

What is ARR5 and why are antibodies against it important for plant research?

ARR5 is a type-A Arabidopsis Response Regulator that functions as a negative regulator in cytokinin signaling pathways. The protein is rapidly upregulated transcriptionally in response to cytokinin exposure and plays a crucial role in plant development and stress responses.

Antibodies against ARR5 are valuable research tools that enable:

• Detection and quantification of ARR5 protein levels in plant tissues

• Monitoring changes in ARR5 abundance in response to various stimuli

• Immunoprecipitation experiments to identify ARR5-interacting proteins

• Visualization of ARR5 subcellular localization

These applications are fundamental to understanding cytokinin signaling mechanisms, which influence numerous aspects of plant growth, development, and stress responses .

How does ARR5 function in cytokinin signaling pathways?

ARR5 functions as a negative regulator in cytokinin signaling through a phosphorelay mechanism. The functional pathway operates as follows:

• Cytokinin binding activates histidine kinase receptors (AHKs)

• Phosphate groups are transferred through Arabidopsis Histidine Phosphotransfer proteins (AHPs)

• AHPs transfer phosphate to the conserved aspartate residue (D87) in the receiver domain of ARR5

• Phosphorylated ARR5 becomes active and negatively regulates cytokinin responses

Research using ARR5 antibodies has demonstrated that this protein's function is strictly dependent on receiver domain phosphorylation, as mutation of the phosphorylation site (D87A) renders the protein non-functional in complementation assays. Additionally, experiments with phosphomimic mutants (D87E) show partial functionality, indicating that the phosphorylated state is the active form .

What are the key considerations when selecting an ARR5 antibody for research?

When selecting an ARR5 antibody for plant research, researchers should consider:

• Specificity: The antibody should specifically recognize ARR5 without cross-reactivity to other type-A ARRs (ARR3, ARR4, ARR6, etc.) which share sequence similarity. Validation through Western blots using knockout mutants is essential.

• Application compatibility: Verify the antibody has been validated for your specific application (Western blot, immunoprecipitation, immunofluorescence, etc.).

• Species reactivity: Most ARR5 antibodies are developed against Arabidopsis thaliana. If working with other plant species, cross-reactivity should be validated.

• Recognition of modifications: Consider whether the antibody can detect phosphorylated forms of ARR5, which may be crucial for studying activation states.

• Epitope location: Antibodies targeting different regions may yield different results, especially if ARR5 undergoes proteolytic processing or conformational changes .

How can ARR5 antibodies be used to study protein degradation dynamics in cytokinin signaling?

ARR5 protein degradation dynamics can be effectively studied using specific antibodies through several methodological approaches:

• Cycloheximide chase assays: ARR5 antibodies enable tracking of protein degradation rates after blocking protein synthesis with cycloheximide. This approach has revealed that ARR5 is rapidly degraded (half-life of approximately 30 minutes) in the absence of cytokinin but stabilized when cytokinin is present.

• Proteasome inhibitor studies: Combining ARR5 antibody detection with proteasome inhibitors (MG132) helps determine if degradation occurs via the ubiquitin-proteasome pathway.

• Phosphorylation-dependent stability: Using ARR5 antibodies in conjunction with phosphorylation site mutants (D87A, D87E) reveals how phosphorylation state affects protein stability.

Research using these approaches has demonstrated that cytokinin treatment significantly delays ARR5 turnover, stabilizing the protein within 30 minutes of application and showing sensitivity to cytokinin concentrations as low as 10 nM. Importantly, this stabilization is dependent on an intact AHK-AHP phosphorelay system, as cytokinin fails to stabilize ARR5 in ahk3,4 and ahp1,2,3,4 mutant backgrounds .

What techniques can be used to distinguish between phosphorylated and non-phosphorylated forms of ARR5?

Distinguishing between phosphorylated and non-phosphorylated ARR5 requires specialized techniques:

• Phospho-specific antibodies: Antibodies specifically raised against phosphorylated D87 of ARR5 can directly detect the active form. These require careful validation with phosphorylation-site mutants (D87A).

• Phos-tag SDS-PAGE: This modified gel electrophoresis technique incorporates Phos-tag molecules that bind phosphorylated proteins, causing a mobility shift that can be detected with standard ARR5 antibodies.

• 2D gel electrophoresis: Combining isoelectric focusing with SDS-PAGE followed by ARR5 immunoblotting separates phosphorylated forms based on charge differences.

• Mass spectrometry after immunoprecipitation: ARR5 antibodies can be used to purify the protein, followed by mass spectrometry analysis to identify and quantify phosphorylated peptides.

• In vivo phosphorylation assays: Combined with ARR5 antibodies for immunoprecipitation, radioactive phosphate (³²P) labeling can track phosphorylation dynamics in response to cytokinin.

Research using these methods has demonstrated that the phosphorylation status of ARR5 at D87 is critical for its function in cytokinin signaling pathways .

How can ARR5 antibodies be used to investigate cross-talk between cytokinin and other hormonal signaling pathways?

ARR5 antibodies offer powerful tools for investigating hormonal cross-talk through several sophisticated approaches:

• Co-immunoprecipitation studies: ARR5 antibodies can pull down protein complexes in plants treated with multiple hormones, revealing interaction partners that may serve as nodes between signaling networks.

• ChIP-seq analysis: For ARR5-associated transcription factors, chromatin immunoprecipitation with ARR5 antibodies followed by sequencing can identify genomic regions influenced by multiple hormone pathways.

• Proximity labeling: Combining ARR5 antibodies with techniques like BioID or APEX2 allows identification of proteins in close proximity to ARR5 under different hormonal treatments.

• Protein stability assays: ARR5 antibodies enable monitoring of how different hormones affect ARR5 stability, revealing post-translational regulation mechanisms.

• Phosphorylation dynamics: Using phospho-specific antibodies against ARR5 can demonstrate how other hormones influence its activation state.

These approaches have revealed interactions between cytokinin signaling and other pathways including auxin, ethylene, and abscisic acid signaling networks, with ARR5 serving as an integration point for multiple hormonal inputs .

What are the optimal protocols for using ARR5 antibodies in Western blotting of plant samples?

Optimized Western Blotting Protocol for ARR5 Detection:

• Sample preparation:

  • Harvest plant tissue and flash-freeze in liquid nitrogen

  • Grind tissue to a fine powder and extract proteins in buffer containing:

    • 50 mM Tris-HCl pH 7.5

    • 150 mM NaCl

    • 1 mM EDTA

    • 10% glycerol

    • 1% Triton X-100

    • 1 mM DTT

    • Protease inhibitor cocktail

    • Phosphatase inhibitors (10 mM NaF, 1 mM Na₃VO₄)

  • Centrifuge at 14,000g for 15 minutes at 4°C

  • Collect supernatant and quantify protein concentration

• Gel electrophoresis:

  • Use 12% SDS-PAGE gels for optimal ARR5 resolution

  • Load 30-50 μg total protein per lane

  • Include phosphorylation-deficient (D87A) controls

• Transfer conditions:

  • Transfer to PVDF membrane (0.45 μm) at 100V for 1 hour in cold transfer buffer

  • For phosphorylated ARR5 detection, add phosphatase inhibitors to transfer buffer

• Antibody incubation:

  • Block membrane with 5% non-fat dry milk in TBST for 1 hour

  • Dilute primary ARR5 antibody 1:1000 in 5% BSA in TBST

  • Incubate overnight at 4°C with gentle agitation

  • Wash 3× with TBST for 10 minutes each

  • Incubate with HRP-conjugated secondary antibody (1:5000) for 1 hour at room temperature

  • Wash 3× with TBST for 10 minutes each

• Detection:

  • Use enhanced chemiluminescence substrate

  • Expected ARR5 band size: approximately 25 kDa (native) or 28 kDa (myc-tagged)

This protocol has been optimized based on experimental conditions used in studies of ARR5 protein stability and phosphorylation status .

How can immunoprecipitation with ARR5 antibodies be optimized to identify interaction partners?

Optimized Immunoprecipitation Protocol for ARR5 Interaction Partners:

• Sample preparation:

  • Cross-link plant tissue with 1% formaldehyde for 10 minutes under vacuum

  • Quench with 125 mM glycine for 5 minutes

  • Extract proteins using buffer containing:

    • 50 mM HEPES pH 7.5

    • 150 mM NaCl

    • 1 mM EDTA

    • 1% Triton X-100

    • 0.1% sodium deoxycholate

    • 0.1% SDS

    • 1 mM PMSF

    • Protease inhibitor cocktail

    • Phosphatase inhibitors

• Pre-clearing:

  • Incubate lysate with protein A/G beads and non-immune IgG for 1 hour at 4°C

  • Remove beads by centrifugation

• Immunoprecipitation:

  • Add ARR5 antibody (5 μg per 1 mg protein lysate)

  • Incubate overnight at 4°C with gentle rotation

  • Add pre-washed protein A/G beads and incubate for 3 hours

  • Wash beads 4× with wash buffer of decreasing stringency

• Elution and analysis:

  • Elute proteins with 0.1 M glycine pH 2.5 or by boiling in SDS sample buffer

  • For mass spectrometry analysis, digest eluted proteins with trypsin

  • For Western blot validation, probe for specific suspected interaction partners

• Controls:

  • Include wild-type vs. arr5 mutant tissues

  • Conduct parallel IPs with non-immune IgG

  • Compare cytokinin-treated vs. untreated samples

  • Include phosphorylation site mutants (D87A, D87E) for phosphorylation-dependent interactions

This methodology has been successfully used to identify components of the cytokinin signaling pathway that interact with ARR5, including connections to the AHK-AHP phosphorelay system .

What are the best approaches for using ARR5 antibodies in immunolocalization studies?

Optimized Immunolocalization Protocol for ARR5:

• Sample preparation:

  • Fix plant tissues in 4% paraformaldehyde in PBS for 1 hour under vacuum

  • Embed in paraffin or prepare for cryosectioning

  • Section tissues at 8-10 μm thickness

  • For whole-mount preparations, use seedlings with cell walls permeabilized by enzyme treatment

• Antigen retrieval:

  • Perform antigen retrieval by heating sections in 10 mM sodium citrate buffer (pH 6.0) for 10 minutes

  • This step is critical for detecting native ARR5

• Immunolabeling:

  • Block with 3% BSA, 0.1% Triton X-100 in PBS for 1 hour

  • Incubate with primary ARR5 antibody (1:100-1:200 dilution) overnight at 4°C

  • Wash 3× with PBS, 10 minutes each

  • Incubate with fluorophore-conjugated secondary antibody (1:500) for 2 hours

  • Counterstain nuclei with DAPI (1 μg/ml)

  • Mount in anti-fade medium

• Controls and validation:

  • Include arr5 knockout tissue as negative control

  • Use pre-immune serum or non-specific IgG as antibody control

  • Compare localization patterns with ARR5-GFP fusion proteins

  • For phosphorylation-dependent localization, include samples treated with phosphatase

• Confocal imaging parameters:

  • Use appropriate excitation/emission settings for the secondary antibody fluorophore

  • Capture Z-stacks with 0.5-1 μm step size

  • Process images with minimal adjustments applied equally to experimental and control samples

This approach has revealed that ARR5 localization changes in response to cytokinin treatment, with increased nuclear accumulation occurring within 30 minutes of hormone application, corresponding to the timing of protein stabilization .

Why might Western blots with ARR5 antibodies show unexpectedly low signal or multiple bands?

Troubleshooting Low Signal or Multiple Bands with ARR5 Antibodies:

Research has shown that ARR5 protein has a short half-life (approximately 30 minutes) in the absence of cytokinin, making it particularly challenging to detect in standard Western blots. Additionally, ARR5 exists in both phosphorylated and non-phosphorylated forms, which may appear as a doublet on standard SDS-PAGE gels. The phosphorylated form specifically increases in abundance following cytokinin treatment .

How can specificity of ARR5 antibodies be validated in experimental systems?

Comprehensive ARR5 Antibody Validation Approaches:

• Genetic validation:

  • Test antibody reactivity in arr5 knockout mutants (should show no signal)

  • Test in arr3,4,5,6 quadruple mutants complemented with ARR5 (should restore signal)

  • Compare signal in ARR5 overexpression lines (should show increased signal intensity)

• Biochemical validation:

  • Perform peptide competition assays using the immunizing peptide

  • Test cross-reactivity with recombinant ARR3, ARR4, ARR6 proteins

  • Validate phospho-specific antibodies with phosphatase-treated samples

• Functional validation:

  • Correlate antibody signal with ARR5 transcript levels in cytokinin-induced samples

  • Verify expected molecular weight shifts with epitope-tagged versions (e.g., myc-ARR5)

  • Confirm expected cellular localization pattern

• Application-specific validation:

  • For IP applications, validate recovered proteins by mass spectrometry

  • For ChIP applications, verify enrichment of known ARR5-regulated promoters

  • For immunohistochemistry, compare with fluorescent protein fusions

Research has demonstrated that validating ARR5 antibodies against phosphorylation site mutants (D87A) is particularly important, as this mutation renders ARR5 non-functional in cytokinin signaling but should still be detected by antibodies targeting regions outside the phosphorylation domain .

What strategies can address ARR5 antibody detection challenges in tissues with complex protein composition?

Strategies for Improving ARR5 Detection in Complex Tissue Samples:

• Enrichment techniques:

  • Perform nuclear fractionation to concentrate ARR5 (predominantly nuclear localized)

  • Use immunoprecipitation before Western blotting for signal enrichment

  • Apply size-exclusion chromatography to separate ARR5 from abundant proteins

• Signal enhancement approaches:

  • Pre-treat plants with cytokinin (10-100 nM) for 1-2 hours to stabilize ARR5

  • Add MG132 (proteasome inhibitor) to prevent degradation

  • Use highly sensitive chemiluminescent substrates or fluorescent secondary antibodies

• Background reduction methods:

  • Optimize blocking conditions (5% BSA often superior to milk for phospho-proteins)

  • Test different antibody dilutions and incubation temperatures

  • Increase washing stringency with higher salt or detergent concentrations

• Tissue-specific considerations:

  • For root tissues: enrich for root tips where cytokinin signaling is highly active

  • For shoot apical meristems: use microdissection to isolate tissues with high ARR5 expression

  • For senescent tissues: add additional protease inhibitors to prevent degradation

Research has shown that ARR5 protein levels vary significantly across tissue types and developmental stages, with highest expression in actively dividing regions like root meristems and shoot apical meristems. Additionally, the protein is stabilized in response to cytokinin, making detection more reliable in cytokinin-treated samples .

How can ARR5 antibodies be used in combination with other techniques to study cytokinin signaling dynamics?

Integrated Approaches Combining ARR5 Antibodies with Other Techniques:

• ChIP-seq analysis:

  • Use ARR5 antibodies for chromatin immunoprecipitation followed by sequencing

  • Map genome-wide binding sites of ARR5 in response to cytokinin

  • Compare with transcriptome data to identify direct vs. indirect targets

  • Protocol modifications: include protein-protein crosslinking with DSP before formaldehyde to capture ARR5 interactions with DNA-binding partners

• Proximity labeling proteomics:

  • Generate ARR5-BioID or ARR5-TurboID fusion proteins

  • Validate expression and functionality using ARR5 antibodies

  • Identify proteins in close proximity to ARR5 under different cytokinin conditions

  • This reveals the dynamic ARR5 interactome dependent on cytokinin signaling

• Live-cell imaging combined with immunofluorescence:

  • Track ARR5-FP fusions in live cells for dynamics

  • Fix cells at specific timepoints and perform immunostaining with phospho-specific ARR5 antibodies

  • Correlate protein movement with phosphorylation state

• Single-cell proteomics validation:

  • Use ARR5 antibodies to validate single-cell proteomics data

  • Map cell type-specific ARR5 expression and phosphorylation states

  • Correlate with cytokinin response markers

These integrated approaches have revealed that ARR5 function is highly context-dependent, with its activity and stability regulated by cytokinin through phosphorylation-dependent mechanisms involving the AHK-AHP phosphorelay system .

What quantitative methods can be used to measure ARR5 protein levels and phosphorylation states?

Quantitative Methods for ARR5 Protein Analysis:

For optimal quantification of phosphorylated ARR5, research has demonstrated that a combination of phospho-specific antibodies with phospho-enrichment techniques (such as metal oxide affinity chromatography) followed by mass spectrometry provides the most comprehensive analysis. This approach can detect changes in ARR5 phosphorylation at D87 within minutes of cytokinin application, corresponding to changes in protein stability and function .

How can protein-protein interactions involving ARR5 be studied using antibody-based techniques?

Antibody-Based Methods for Studying ARR5 Protein Interactions:

• Co-immunoprecipitation (Co-IP):

  • Precipitate ARR5 using specific antibodies and identify co-precipitating proteins

  • Enhanced protocol: Use membrane-permeable crosslinkers before lysis to capture transient interactions

  • Validation approach: Perform reverse Co-IP with antibodies against suspected interaction partners

  • Application: Has identified interactions between ARR5 and components of the AHK-AHP phosphorelay system

• Proximity Ligation Assay (PLA):

  • Detect in situ protein interactions at single-molecule resolution

  • Requires primary antibodies against both ARR5 and interaction partner

  • Provides spatial information about where interactions occur within cells

  • Particularly useful for detecting phosphorylation-dependent interactions

• Bimolecular Fluorescence Complementation (BiFC) validation:

  • Express ARR5 fused to half of a fluorescent protein

  • Express potential interaction partner fused to complementary half

  • Validate interaction using ARR5 antibodies in parallel Western blots

  • Assess how mutations in ARR5 phosphorylation site affect interactions

• Protein microarrays with ARR5 antibody detection:

  • Screen proteome-wide arrays with recombinant ARR5

  • Detect interactions using ARR5 antibodies

  • Compare interaction profiles with unphosphorylated vs. phosphorylated ARR5

  • Identify novel interaction partners involved in cytokinin signaling

These methods have revealed that ARR5 interactions are highly dependent on its phosphorylation status, with the D87 phosphorylation site being critical for interactions with downstream signaling components. Additionally, cytokinin treatment alters the ARR5 interactome by stabilizing the protein and modifying its phosphorylation state .