AXR4 Antibody

Definition and Purpose of AXR4 Antibody

AXR4 Antibody is a polyclonal or monoclonal antibody designed to bind specifically to the AXR4 protein. Its primary purpose is to enable precise detection and localization of AXR4 in plant tissues, facilitating research into auxin transport mechanisms, root development, and protein trafficking pathways.

Key Features:

• Target Protein: AXR4 (plant-specific ER accessory protein).

• Applications: Immunolocalization, Western blotting, co-immunoprecipitation (Co-IP), and genetic studies.

• Relevance: Essential for understanding AXR4’s role in auxin influx carrier trafficking, which impacts root gravitropism and nutrient acquisition.

Production and Validation Methods

AXR4 Antibody is typically produced using recombinant protein approaches, as part of larger antibody resource projects (e.g., the Arabidopsis root protein antibody initiative) . Validation involves rigorous testing to ensure specificity and functionality.

Production Pipeline

1. Antigen Design:

   • Identification of immunogenic regions in AXR4 using bioinformatics tools.

   • Avoidance of conserved regions to minimize cross-reactivity (e.g., <40% similarity cutoff) .

2. Expression and Purification:

   • Recombinant AXR4 protein expressed in heterologous systems (e.g., E. coli or insect cells).

   • Affinity purification using Protein A/G or Ni-NTA columns .

Validation Metrics

Notes:

• Western Blot: Detects AXR4 in protein extracts, confirming expression levels.

• Immunolocalization: Visualizes AXR4’s ER localization in root cells .

• Mutant Validation: Absence of signal in axr4 mutants confirms antibody specificity .

Applications in Research

AXR4 Antibody is indispensable for studying AXR4’s interactions, localization, and functional roles.

Key Applications

1. Protein Localization:

   • ER vs. Plasma Membrane (PM): AXR4 localizes to the ER, as confirmed by colocalization with ER markers like BPL1 .

   • Trafficking Defects: In axr4 mutants, AUX1 mislocalizes to the ER, detected via co-immunolocalization with AXR4 Antibody .

2. Interaction Studies:

   • Co-IP Experiments: AXR4 physically interacts with AUX1, demonstrated by pull-down assays in insect cells .

   • Aggregation Prevention: AXR4 reduces AUX1 aggregation in a dose-dependent manner, inferred from SDS-PAGE analysis of co-expressed proteins .

3. Genetic and Physiological Studies:

   • Mutant Phenotyping: axr4 mutants exhibit auxin-resistant root growth and impaired gravitropism, linked to AUX1 mislocalization .

   • Complementation Assays: AXR4-GFP transgenes restore AUX1/LAX2 PM localization in mutants, validated via immunolocalization .

Mechanistic Insights

1. ER Accessory Protein Function:

   • AXR4 prevents AUX1/LAX2 aggregation in the ER, acting as a molecular chaperone .

   • Mutations in AXR4’s α/β hydrolase fold domain (e.g., L140V) do not impair function, suggesting enzymatic activity is unnecessary .

2. Trafficking Specificity:

   • AXR4 selectively regulates AUX1/LAX2 localization, unlike PIN efflux carriers (PIN1/PIN2), which remain unaffected in axr4 mutants .

3. Family-Wide Regulation:

   • AXR4 may regulate the entire AUX1/LAX family, given their high sequence similarity (76–82%) .

Table 1: AXR4 Antibody Performance in Detection Methods

Figure 1: AXR4 Localization in Wild-Type vs. axr4 Mutant

(Hypothetical Representation)