ABCG14 Antibody

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Description

Definition and Role of ABCG14 Antibody

The ABCG14 antibody is a specialized immunological tool developed to detect and study the Arabidopsis thaliana ABCG14 (AtABCG14) protein, a member of the ATP-binding cassette (ABC) transporter family. This antibody is primarily used in plant biology research to investigate ABCG14's role in cytokinin transport, a critical process regulating plant growth and development .

Protein Characteristics

  • Domain Architecture: ABCG14 is a plasma membrane-localized half-transporter requiring homodimerization for activity. It contains a nucleotide-binding domain (NBD) and transmembrane domains (TMDs) typical of ABC transporters .

  • Homodimerization: Co-immunoprecipitation assays confirmed that ABCG14 forms homodimers, essential for its transporter function .

Cytokinin Transport Mechanism

ABCG14 mediates the acropetal (root-to-shoot) translocation of cytokinins, particularly trans-zeatin (tZ) and isopentenyladenine (iP) types. Key findings include:

  • Efflux Activity: ABCG14 exports cytokinins from root pericycle and stelar cells into the xylem, enabling long-distance transport .

  • Substrate Specificity: Biochemical assays demonstrated ABCG14 transports multiple cytokinin species, including tZ-riboside, iP-riboside, and cis-zeatin .

Phenotypic Effects of ABCG14 Knockout

ParameterWild-Typeatabcg14 MutantSource
Shoot Cytokinin LevelsNormalReduced tZ-type cytokinins
Root Cytokinin LevelsNormalElevated tZ- and iP-types
Plant GrowthRobustDwarfism, delayed senescence

Localization and Expression

  • Tissue Specificity: ABCG14 is highly expressed in root pericycle and stele cells, overlapping with cytokinin biosynthesis genes like IPT3 and CYP735A2 .

  • Subcellular Localization: GFP-tagged ABCG14 localized to the plasma membrane, confirmed via co-staining with FM4-64 and propidium iodide .

Transport Assays

  • Radiotracer Studies: Detached leaves overexpressing ABCG14 showed 4x lower accumulation of C¹⁴-labeled tZ compared to wild-type, indicating efflux activity .

  • Vanadate Sensitivity: ABCG14-mediated cytokinin export was inhibited by sodium ortho-vanadate, a ABC transporter inhibitor .

Applications of ABCG14 Antibody in Research

  • Protein Detection: Used in Western blotting to confirm ABCG14 expression in transgenic lines (e.g., GFP- or Myc-tagged constructs) .

  • Subcellular Localization: Facilitated visualization of ABCG14’s plasma membrane localization via fluorescence microscopy .

  • Mechanistic Studies: Enabled validation of ABCG14 homodimerization through co-immunoprecipitation .

Implications for Plant Biology

ABCG14 is pivotal for maintaining cytokinin homeostasis, influencing:

  • Root Development: Mutants exhibit reduced sensitivity to exogenous cytokinins due to elevated root cytokinin levels .

  • Stress Responses: ABCG14-mediated transport may modulate drought and nutrient stress resilience via cytokinin signaling .

Outstanding Questions and Future Directions

  • Dimerization Partners: While ABCG14 forms homodimers, potential heterodimer interactions remain unexplored .

  • Crosstalk with Other Transporters: Synergy with purine permeases (PUPs) or equilibrative nucleoside transporters (ENTs) requires investigation .

Product Specs

Buffer
Preservative: 0.03% ProClin 300; Constituents: 50% Glycerol, 0.01M PBS, pH 7.4
Form
Liquid
Lead Time
Made-to-order (14-16 weeks)
Synonyms
ABCG14; INT211; WBC14; At1g31770; F27M3.2; F5M6.22; ABC transporter G family member 14; ABC transporter ABCG.14; AtABCG14; Protein INSENSITIVE TO TEMPERATURE 211; White-brown complex homolog protein 14; AtWBC14
Target Names
ABCG14
Uniprot No.

Target Background

Function
This antibody targets ABCG14, a positive regulator of plant growth functioning as an efflux pump. It is involved in the primary long-distance transport of cytokinins (CKs) from roots to shoots (acropetal transport) via the xylem sap. In conjunction with ABCG9 and ABCG11, ABCG14 is crucial for vascular development by regulating lipid/sterol homeostasis. Furthermore, it plays a role in CK-dependent responses to oxidative stress, such as that induced by hydrogen peroxide (H₂O₂). In the context of microbial infection, ABCG14 is required for the SNC1-mediated defense response against the virulent pathogen *Pseudomonas syringae* pv. tomato DC3000 by facilitating the accumulation of trans-zeatin (tZ)-type cytokinins in the shoot.
Gene References Into Functions
  • ABCG14 mediates cytokinin (CK) transport from roots to shoots. Impairment of ABCG14 results in a deficiency of trans-zeatin (tZ)-type CKs in the shoot, thereby suppressing the SNC1-mediated defense response. (PMID: 28398838)
  • ABCG14 is essential for the acropetal translocation of root-synthesized cytokinins. AtABCG14 knockout significantly impairs cytokinin translocation from roots to shoots, affecting plant growth and development. (PMID: 24513716)
  • Studies demonstrate that AtABCG14 is critical for cytokinin translocation to the shoot. (PMID: 24778257)
Database Links

KEGG: ath:AT1G31770

STRING: 3702.AT1G31770.1

UniGene: At.22580

Protein Families
ABC transporter superfamily, ABCG family, Eye pigment precursor importer (TC 3.A.1.204) subfamily
Subcellular Location
Cell membrane; Multi-pass membrane protein.
Tissue Specificity
Accumulates primarily in the pericycle and stelar cells of roots. Expressed in leaves, stems, flowers and siliques, and, at low levels, in roots. Accumulates in the phloem.

Q&A

What is ABCG14 and why is it important in plant research?

ABCG14 (ATP-binding cassette transporter subfamily G14) is a plasma membrane-localized transporter protein in Arabidopsis thaliana that forms homodimers and functions in the transport of cytokinins, primarily trans-zeatin (tZ) and isopentenyladenine (iP) types. It plays a crucial role in plant growth and development by facilitating the acropetal (root to shoot) translocation of root-synthesized cytokinins . Research has demonstrated that knocking out AtABCG14 impairs the distribution of root-synthesized tZ- and DHZ-type cytokinins between root and shoot tissues, leading to significant developmental defects . This makes ABCG14 a key component for understanding hormone transport mechanisms and long-distance signaling in plants.

How can I verify the subcellular localization of ABCG14 using antibodies?

To verify ABCG14 subcellular localization, researchers typically employ a multi-faceted approach:

  • Generate GFP-ABCG14 fusion constructs (e.g., 35S::EGFP-ABCG14)

  • Express the fusion protein in Arabidopsis leaf protoplasts or stable transformants

  • Perform direct fluorescence microscopy and immunofluorescence with anti-GFP antibodies

  • Conduct co-localization studies with plasma membrane markers like FM4-64

  • Confirm localization using plasmolysis experiments to distinguish between plasma membrane and cell wall localization

  • Validate by subcellular fractionation and Western blotting

Research has confirmed that ABCG14 primarily localizes to the plasma membrane, with fluorescence signals appearing at the rims of protoplasts and colocalizing with FM4-64 staining but separating from propidium iodide-stained cell walls during plasmolysis . Aqueous-polymer two-phase partitioning followed by immunoblotting with anti-GFP antibodies has further demonstrated ABCG14's presence in the plasma membrane fraction .

What controls should I include when using antibodies to detect ABCG14 in Western blots?

When performing Western blots to detect ABCG14, include the following essential controls:

Control TypePurposeExample
Positive controlConfirms antibody functionalityExtract from ABCG14 overexpression lines
Negative controlEstablishes specificityExtract from abcg14 knockout mutants
Loading controlEnsures equal loadingProbing with anti-actin antibodies
Membrane fraction markerConfirms proper fractionationAnti-H⁺-ATPase antibody for plasma membrane fraction
Organelle markersExcludes contaminationAnti-vacuolar H⁺-pyrophosphatase for tonoplast
Size verificationConfirms expected molecular weightMolecular weight standards

For membrane proteins like ABCG14, proper extraction and sample preparation are critical. Studies have successfully used aqueous-polymer two-phase partitioning to enrich plasma membrane fractions, followed by detection with antibodies against fusion tags like GFP .

How do expression patterns of ABCG14 vary across plant tissues and developmental stages?

ABCG14 shows distinct tissue-specific expression patterns:

  • Highest expression occurs in root tissues, particularly in the pericycle cells

  • Expression is detectable but lower in shoots and reproductive tissues

  • Within the root, expression is strongest in the vascular cylinder

  • In leaves, expression is restricted primarily to the vascular tissues

For accurate expression analysis, a combination of approaches is recommended:

  • qRT-PCR using gene-specific primers with ACTIN2 or UBIQUITIN10 as reference genes

  • Promoter-reporter fusions (ABCG14::GUS or ABCG14::EGFP) for tissue-specific localization

  • Immunohistochemistry with specific antibodies for protein-level detection

Understanding these expression patterns is crucial for interpreting ABCG14 function in cytokinin transport .

What are the recommended methods for generating and validating ABCG14 fusion proteins for antibody detection?

Generation and validation of ABCG14 fusion proteins should follow these methodological steps:

  • Construct Design:

    • Create N-terminal fusions (e.g., 35S::EGFP-ABCG14) or C-terminal fusions based on experimental needs

    • Include both constitutive (35S) and native promoter (ABCG14 promoter) versions

    • Use gateway cloning systems with appropriate entry and destination vectors

  • Vector Construction Protocol:

    • Amplify the ABCG14 coding region using specific primers (e.g., ABCG14-P3 and ABCG14-P4)

    • Clone into gateway entry vectors (e.g., pDONR207) via BP reaction

    • Transfer to destination vectors (e.g., pMDC43 for GFP fusions) via LR reaction

  • Validation:

    • Confirm functionality through complementation tests in abcg14 mutants

    • Verify protein expression by Western blotting with anti-tag antibodies

    • Test subcellular localization using microscopy

    • Assess transport activity using radiotracer experiments

Researchers have successfully used EGFP-ABCG14 fusions that complement abcg14 mutant phenotypes, demonstrating that the fusion proteins retain biological activity .

How can I optimize immunoprecipitation protocols for studying ABCG14 protein interactions?

For effective immunoprecipitation of ABCG14 complexes:

  • Membrane Protein Extraction:

    • Use nitrogen grinding followed by buffer extraction with protease inhibitors

    • Include membrane-solubilizing detergents (e.g., 1% digitonin or 0.5-1% NP-40)

    • Perform extractions at 4°C to prevent protein degradation

  • Immunoprecipitation Strategy:

    • For GFP-tagged ABCG14, use GFP-Trap beads or anti-GFP antibodies coupled to protein A/G

    • Pre-clear lysates to reduce non-specific binding

    • Include appropriate controls (untransformed plants, unrelated membrane protein fusions)

  • Interaction Verification:

    • Perform reciprocal co-immunoprecipitation experiments

    • Validate interactions with alternative methods (yeast two-hybrid, BiFC)

    • Consider chemical crosslinking to stabilize transient interactions

Since ABCG14 forms homodimers , tagged versions can be used to study dimerization dynamics and potential interactions with other transporters or regulatory proteins.

What techniques are most effective for quantifying ABCG14 expression changes in response to cytokinin treatments?

To quantify ABCG14 expression changes after cytokinin treatment:

MethodApplicationStrengthsConsiderations
qRT-PCRTranscript levelsHigh sensitivity, quantitativePrimer design: ABCG14-P13/P14 with ACTIN2 or UBIQUITIN10 references
Western blotProtein levelsDirect protein measurementMembrane fraction isolation required
Reporter assaysSpatial regulationVisual tissue distributionMay not reflect post-transcriptional regulation
Radioisotope transportFunctional activityMeasures actual transportRequires C14-tZ and careful experimental design

Research has shown that ABCG14 expression can be induced by certain cytokinins, particularly in cytokinin-deficient backgrounds . When designing cytokinin treatment experiments, include appropriate concentration gradients (1-100 nM) and time-course measurements (0.5-24 hours) to capture both rapid and sustained responses.

How should I design experiments to distinguish between ABCG14 transport of different cytokinin species?

Designing experiments to characterize ABCG14 substrate specificity:

  • Radioisotope Transport Assays:

    • Use C14-labeled cytokinins (tZ, iP) in uptake/efflux experiments

    • Compare transport rates between wild-type, abcg14 mutants, and complemented lines

    • Include specific inhibitors (e.g., sodium ortho-vanadate) to confirm ABC transporter involvement

  • Cytokinin Profiling:

    • Perform LC-MS/MS analysis of cytokinin species in roots and shoots

    • Compare profiles between wild-type, abcg14 mutants, and ABCG14 overexpression lines

    • Quantify individual cytokinin species and their derivatives to assess transport specificity

  • Grafting Experiments:

    • Perform reciprocal grafting between wild-type and abcg14 mutants

    • Analyze cytokinin content in scions and rootstocks

    • Determine which cytokinin species require ABCG14 for long-distance transport

Research has demonstrated that ABCG14 is essential for tZ-type cytokinin translocation, while its role in iP-type transport has been more recently established .

How do I interpret contradictory results between transcript levels and protein abundance of ABCG14?

When facing discrepancies between ABCG14 mRNA and protein levels:

  • Consider post-transcriptional regulation:

    • Evaluate microRNA-mediated regulation

    • Assess mRNA stability differences across conditions

    • Examine translation efficiency factors

  • Investigate protein stability factors:

    • Test if protein degradation rates vary between conditions

    • Check for post-translational modifications affecting stability

    • Consider membrane protein turnover mechanisms

  • Reconciliation approaches:

    • Perform time-course analyses to identify temporal shifts between transcript and protein changes

    • Use cycloheximide or MG132 to block protein synthesis or degradation, respectively

    • Implement pulse-chase experiments to determine protein half-life

  • Validation strategies:

    • Create translational reporter fusions

    • Use polysome profiling to assess translation efficiency

    • Apply ribosome profiling techniques

Plasma membrane proteins like ABCG14 often show complex regulation patterns that may not correlate directly with transcript levels due to trafficking, membrane insertion, and turnover processes.

What are the most common challenges in ABCG14 antibody-based assays and how can they be overcome?

Common challenges in ABCG14 antibody assays:

ChallengeCauseSolution
Low signalInsufficient extraction efficiencyUse optimized membrane protein extraction methods
Multiple bandsDegradation productsInclude protease inhibitor cocktail; keep samples cold
High backgroundNon-specific bindingOptimize blocking buffers; increase washing stringency
Poor reproducibilityVariability in membrane fraction purityInclude membrane marker controls (H⁺-ATPase)
Inconsistent loadingDifficult standardization for membrane proteinsNormalize to membrane-specific markers rather than total protein

Researchers have successfully used two-phase partitioning to enrich plasma membrane fractions, which significantly improves detection of ABCG14 . For immunolocalization experiments, optimizing fixation conditions is critical to preserve epitope accessibility while maintaining membrane structure.

How can I validate that my detected ABCG14 signal represents functional protein?

To validate functional ABCG14 detection:

  • Genetic validation:

    • Compare signals between wild-type, knockout mutants, and complemented lines

    • Correlate signal intensity with phenotypic rescue in complementation experiments

  • Biochemical validation:

    • Perform ATP binding/hydrolysis assays to confirm activity

    • Use vanadate-sensitive transport assays with radioisotope-labeled cytokinins

  • Functional correlation:

    • Assess cytokinin distribution in plants with varying ABCG14 levels

    • Perform reciprocal grafting experiments between wild-type and mutant plants

    • Correlate protein levels with transport activity

  • Structure-function analysis:

    • Create point mutations in key functional domains and assess both detection and activity

    • Compare wild-type and mutant protein localization patterns

Research has demonstrated that ABCG14 functions as an exporter, with transgenic plants overexpressing ABCG14 showing significantly reduced cellular retention of radiolabeled tZ compared to wild-type controls .

What approaches can resolve discrepancies in ABCG14 subcellular localization data?

When facing conflicting localization data:

  • Technical reconciliation:

    • Use multiple independent localization methods (fluorescent protein fusions, immunolocalization, subcellular fractionation)

    • Verify fusion protein functionality through complementation tests

    • Employ high-resolution microscopy techniques (STED, SIM)

  • Biological considerations:

    • Investigate potential conditional localization (stress, developmental stage)

    • Examine tissue-specific differences in targeting

    • Consider polarized distribution within the plasma membrane

  • Validation approaches:

    • Perform co-localization with multiple membrane markers

    • Use plasmolysis experiments to distinguish membrane from cell wall signals

    • Apply biochemical fractionation with quantitative assessment of distributions

Research with ABCG14 has successfully combined fluorescence microscopy, membrane marker co-localization, plasmolysis experiments, and biochemical fractionation to confidently establish its plasma membrane localization .

How might new antibody technologies advance our understanding of ABCG14 conformational changes during transport?

Emerging antibody technologies for ABCG14 research:

  • Conformation-specific antibodies:

    • Develop antibodies targeting ATP-bound, nucleotide-free, or substrate-bound states

    • Use these to trap ABCG14 in specific conformational states

    • Correlate conformational distributions with transport activity

  • Nanobody applications:

    • Engineer nanobodies against specific ABCG14 domains

    • Use for super-resolution imaging of native protein

    • Develop conformational biosensors for live-cell imaging

  • Proximity labeling approaches:

    • Employ antibody-enzyme fusions (APEX, BioID) to identify proximal proteins

    • Map the ABCG14 interactome during active transport

    • Identify regulatory proteins that modulate activity

These approaches could help resolve outstanding questions about how ABCG14 binds and transports different cytokinin species, potentially explaining its dual role in tZ and iP transport .

What experimental strategies could clarify the relationship between ABCG14 dimerization and transport activity?

To investigate ABCG14 dimerization and function:

  • Structural analysis:

    • Generate structural models based on related ABC transporters

    • Identify putative dimerization interfaces

    • Design targeted mutations to disrupt dimerization

  • Interaction dynamics:

    • Apply FRET/FLIM techniques with differentially tagged ABCG14 variants

    • Perform bimolecular fluorescence complementation to visualize dimers in vivo

    • Use co-immunoprecipitation with differently tagged versions to quantify dimerization

  • Functional correlation:

    • Correlate dimerization efficiency with transport activity

    • Create chimeric proteins with heterologous dimerization domains

    • Assess transport of different cytokinin species by various dimeric forms

  • Single-molecule approaches:

    • Apply single-molecule tracking to monitor ABCG14 dynamics in membranes

    • Assess oligomeric state using step-wise photobleaching

    • Correlate mobility with functional states

Research has established that ABCG14 forms homodimers in multiple expression systems, including human HEK293T cells, tobacco, and Arabidopsis , but the functional significance of dimerization for specific cytokinin transport remains to be fully characterized.

How can systems biology approaches integrating ABCG14 antibody data advance our understanding of cytokinin transport networks?

Systems biology strategies for ABCG14 research:

  • Multi-omics integration:

    • Correlate ABCG14 protein levels with cytokinin profiles across tissues

    • Integrate ABCG14 interactome data with transcriptional networks

    • Model cytokinin fluxes based on transporter distribution

  • Spatial mapping:

    • Create high-resolution maps of ABCG14 distribution and abundance

    • Correlate with cytokinin response markers and sensors

    • Develop computational models of hormone gradients

  • Network analysis:

    • Identify regulatory nodes controlling ABCG14 expression and activity

    • Map connections between ABCG14 and other hormone transporters

    • Model crosstalk between cytokinin transport and other signaling pathways

  • Synthetic biology applications:

    • Engineer altered ABCG14 expression/localization patterns

    • Create cytokinin transport circuits with predictable outputs

    • Design synthetic regulatory systems for ABCG14 activity

These approaches could help explain how ABCG14 contributes to the homeostatic mechanisms that compensate for perturbations in synthesis and distribution of different cytokinin types .

What are the emerging methodologies for studying ABCG14 orthologs across plant species?

For cross-species ABCG14 research:

MethodologyApplicationConsiderations
Comparative genomicsIdentify orthologs in non-model speciesFocus on conserved functional domains and motifs
Cross-reactive antibodiesDetect ABCG14-like proteins in diverse speciesDesign against highly conserved epitopes
Heterologous expressionFunctional characterizationExpress candidate orthologs in Arabidopsis abcg14 background
CRISPR-based approachesCreate equivalent mutations across speciesTarget conserved functional residues
Transport assaysCompare substrate specificityStandardize assay conditions across species

Understanding ABCG14 orthologs could reveal how cytokinin transport mechanisms evolved across plant lineages and potentially identify specialized adaptations in different species. Comparative studies may also provide insights into structure-function relationships that are not evident from studying Arabidopsis alone.

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