PIN3 Antibody
Description
Definition and Biological Context of PIN3 Antibody
The PIN3 antibody is a specialized immunological tool designed to detect and study the PIN-FORMED 3 (PIN3) protein, a key auxin efflux carrier in plants. PIN3 regulates polar auxin transport, which governs developmental processes such as gravitropism, shade avoidance, and organogenesis in Arabidopsis thaliana . This antibody enables researchers to investigate PIN3's subcellular localization, expression dynamics, and functional interactions through techniques like immunolocalization, Western blotting, and chromatin immunoprecipitation (ChIP) .
Antibody Generation
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Immunogen: A recombinant protein corresponding to a unique 150-amino-acid sequence in the hydrophilic loop of PIN3 was used for immunization .
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Affinity Purification: Crude antisera were purified using immobilized recombinant PIN3 protein to enhance specificity and signal-to-noise ratio .
Validation Metrics
Auxin Transport Regulation
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Gravitropism: PIN3 antibody revealed asymmetric relocalization of PIN3 in root columella cells within 2–10 minutes of gravistimulation, facilitating auxin redistribution .
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Shade Avoidance: Low red-to-far-red (R:FR) light induces lateral PIN3 relocalization in hypocotyl endodermal cells, enhancing auxin transport to epidermal layers .
Developmental Studies
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Ovule Initiation: PIN3 antibody demonstrated PIN3 accumulation in ovule primordia, with mutants showing a 30–50% reduction in late ovule initiation .
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Lateral Root Formation: PIN3 expression in lateral root primordia depends on ARF7 and FLP transcription factors, as shown by ChIP-PCR .
Phosphorylation-Dependent PIN3 Dynamics
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Phosphomimetic Mutants: Substituting PIN3 serines (S226, S243, S283) with aspartic acid (P1D/P2D) inhibited gravity-induced PIN3 relocation by 60–80% .
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Auxin Feedback: PIN3 expression and localization are upregulated by auxin itself, creating a feed-forward loop during shade avoidance .
Functional Redundancy
Technical Considerations and Limitations
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Cross-Reactivity: Anti-PIN3 exhibits minimal cross-reactivity with PIN1, PIN2, or PIN4 due to epitope specificity .
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Artifact Risks: Prolonged fixation or excessive Brefeldin A treatment causes PIN3 mislocalization to endosomal compartments .
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Quantitative Limits: Western blotting requires ≥10 µg of total protein extract for reliable detection .
Product Specs
Constituents: 50% Glycerol, 0.01M PBS, pH 7.4
Target Background
PIN3's function is extensively documented, with roles in various auxin-mediated processes. Key findings include:
- Gravity Response: PIN3 directs auxin flow to the lower hypocotyl side, promoting growth, and subsequently polarizes to the opposite side, terminating bending. (PMID: 27773568)
- Transcriptional Regulation: Protein kinase CK2 and salicylic acid maintain PIN3 transcriptional expression. (PMID: 24547808)
- Phototropism: Asymmetric auxin distribution, regulated by blue-light-induced PIN3 polarization, is essential for root negative phototropism. (PMID: 23888933)
- Lateral Root Emergence: Sequential induction of PIN3 (auxin efflux) and LAX3 (auxin influx) carriers regulates lateral root emergence. (PMID: 24150423)
- Gravitropic Response: PIN3 is required for the asymmetric auxin distribution underlying the gravitropic response. (PMID: 21569134)
- Guard Cell Polarity: PIN3 polarity in Arabidopsis guard cells is determined by its hydrophilic loop phosphorylation. (PMID: 22902703)
- Lateral Root Initiation: PIN3 is crucial for timely lateral root initiation, particularly within the endodermis. (PMID: 23178590)
- Phototropism: PIN3-mediated auxin gradients participate in pulse-induced phototropism. (PMID: 22843667)
- Phosphorylation and Function: Mutations in phosphorylation sites disrupt PIN3's auxin efflux activity, auxin maxima formation, root growth, and gravitropism. (PMID: 22519832)
- Light-Induced Polarization: Light polarizes PIN3 localization in hypocotyl endodermis cells, affecting auxin distribution and differential growth. (PMID: 21394084)
- Fitness: pin3 mutants exhibit reduced fitness compared to wild-type plants. (PMID: 21149713)
- Cold Stress: Cold stress inhibits PIN3's lateral relocalization, blocking the early gravity response. (PMID: 20040541)
- Cell-Type Specific Expression: NOV is required for cell type-specific PIN3 expression in the root. (PMID: 19880797)
- Gravity Signal Transduction: ARL2 and ARG1 function with PIN3 in a gravity signal transduction pathway within statocytes. (PMID: 18047472)
