ABCG43 Antibody

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Description

Antigen 43 (Ag43) Antibodies

Ag43 is a bacterial autotransporter protein produced by Escherichia coli and other Gram-negative pathogens. It facilitates cell aggregation and biofilm formation through a unique β-helical structure that enables Velcro-like intercellular adhesion .

Key Functional Features:

PropertyDescription
StructureL-shaped β-helix with α<sub>43</sub> passenger domain (~10 nm protrusion)
RoleVirulence factor in urinary/respiratory infections; promotes bacterial aggregation
Phase VariationExpression regulated by phase-variable flu gene

Antibody Applications:

  • Mechanistic Studies: Used to investigate biofilm formation in UPEC strains .

  • Diagnostic Potential: Detects Ag43-expressing bacterial colonies via immunofluorescence .

Growth-Associated Protein 43 (GAP-43) Antibodies

GAP-43 is a neuron-specific phosphoprotein critical for axonal growth, synaptic plasticity, and neural regeneration. Commercial antibodies are widely used in neuroscience research.

Research Findings:

  • Neurodevelopmental Role:
    GAP-43 knockdown in cortical neurons reduces dendritic arborization and disrupts interhemispheric axon elongation .

    • Dendritic branch points: ↓40% in GAP-43-deficient neurons

    • Axon density in corpus callosum: ↓55% compared to controls

  • Cancer Research:
    Overexpression in colorectal cancer cells upregulates ABC transporters (p = 0.001) and downregulates ribosomal biosynthesis pathways (p < 1e-04) .

Comparative Analysis

FeatureAg43 AntibodiesGAP-43 Antibodies
Primary UseBacterial pathogenesis studiesNeuroscience/cancer research
Key Commercial SourcesSanta Cruz Biotechnology Abcam , Cell Signaling
Disease RelevanceUrinary tract infections Neurodevelopmental disorders

Experimental Validation Data

GAP-43 Antibody Specificity (ab75810):

  • Western Blot: Detects 48 kDa band in human/mouse brain lysates .

  • Immunohistochemistry: Strong signal in cortical layers II/III (mouse cerebrum) .

  • Knockdown Validation: RNAi reduces axonal density by 55% (rescued by GAP-43R) .

Limitations:

  • Clone EP890Y fails to detect GAP-43 in PC-12 cells .

  • Ag43 antibodies show cross-reactivity with other AIDA-I autotransporters .

Product Specs

Buffer
Preservative: 0.03% ProClin 300; Constituents: 50% Glycerol, 0.01M PBS, pH 7.4
Form
Liquid
Lead Time
14-16 week lead time (made-to-order)
Synonyms
ABCG43 antibody; PDR15 antibody; At4g15236 antibody; FCAALL.461ABC transporter G family member 43 antibody; ABC transporter ABCG.43 antibody; AtABCG43 antibody; Pleiotropic drug resistance protein 15 antibody
Target Names
ABCG43
Uniprot No.

Target Background

Function
Putative general defense protein.
Database Links
Protein Families
ABC transporter superfamily, ABCG family, PDR (TC 3.A.1.205) subfamily
Subcellular Location
Membrane; Multi-pass membrane protein.

Q&A

What is ABCG43 and what is its known function in Arabidopsis thaliana?

ABCG43 (At4g15236) is an ABC transporter gene in Arabidopsis thaliana whose function has been linked to root-substrate adhesion. Research using T-DNA insertion mutants (abcg43-1, abcg43-2, and abcg43-3) has demonstrated that ABCG43 plays a significant role in how plant roots interact with their growth medium . Plants with ABCG43 mutations showed significantly enhanced root-gel adhesion compared to wild-type plants, with detachment risks 0.25, 0.36, and 0.35 times that of wild-type plants for the three mutant alleles respectively . This suggests ABCG43 may be involved in regulating compounds that affect root adhesion properties, possibly through its role as a transporter.

How were ABCG43 mutants initially identified and characterized?

ABCG43 mutants were identified through a high-throughput centrifuge-based screening method designed to identify novel genes involved in root-substrate adhesion. Initially, the abcg43-1 line was identified from a pooled set of 100 SALK T-DNA insertion lines (stock number N75206) . The screening process involved:

  • Growing seedlings on sterile gel medium

  • Subjecting them to increasing centrifugal forces

  • Identifying plants that remained attached at high centrifugal speeds

  • Self-fertilizing these plants to obtain progeny

  • Confirming the phenotype in subsequent generations

The mutation was confirmed by genomic Next-Generation sequencing followed by Sanger sequencing of T-DNA flanking PCR products . Subsequently, two additional alleles (abcg43-2 and abcg43-3) were identified to confirm the phenotype.

What methods are available for genotyping ABCG43 mutants?

Genotyping ABCG43 mutant alleles can be performed using the following protocol:

  • Extract genomic DNA from 2-week-old plants using a modified Edwards prep method

  • Design T-DNA border and gene-specific primers (available in published supplementary tables)

  • Perform PCR analysis to confirm the genotype

  • For more detailed characterization, PCR products can be purified and extracted from a 1% agarose gel using a QIAquick Gel Extraction Kit

  • Conduct Sanger sequencing using appropriate primers

  • After manual low-quality end trimming, align the sequences to the relevant vector sequence and ABCG43 gene sequence using alignment tools such as MUSCLE

This methodology allows for accurate identification of homozygous and heterozygous plants carrying the mutations.

How can antibodies against ABCG43 be developed for plant research?

Developing antibodies against plant membrane proteins like ABCG43 requires specialized approaches:

  • Antigen Design: Select unique epitopes from ABCG43's extracellular domains or generate peptide antigens from hydrophilic regions of the protein.

  • Expression System Selection: For plant membrane proteins like ABCG43, expression in heterologous systems can be achieved using:

    • Mammalian cell lines like FreeStyle 293 cells, which allow for proper folding and post-translational modifications

    • Plant-based expression systems such as Arabidopsis seeds, which have been shown effective for recombinant antibody production

  • Cloning Strategy: Implement a Golden Gate Cloning approach to generate an Ig dual-expression vector, enabling the linkage of heavy-chain variable and light-chain variable DNA fragments .

  • Screening Method: Employ flow cytometry-based screening to identify high-affinity antibodies, which is significantly faster than conventional cloning-based methods that require sequential steps .

  • Validation: Test antibody specificity using wild-type plants alongside abcg43 mutant alleles as negative controls.

What experimental approaches can be used to study ABCG43's role in root-substrate adhesion?

The centrifuge-based assay described in the literature provides a powerful quantitative method for studying ABCG43's function:

  • Centrifuge Assay Setup:

    • Sow ten seeds onto sterile gel medium in 90mm Petri plates

    • Stack plates vertically (approximately 80°) to encourage roots to grow down the surface

    • Maintain constant light (120-145 μmol m⁻² s⁻¹) at 22°C and 60% relative humidity

  • Adhesion Measurement Protocol:

    • After 5-6 days of growth, subject seedlings to increasing centrifugal forces

    • Place plates in a swing-out-bucket centrifuge in an inverted orientation

    • Apply 1-minute pulses of increasing centrifugal speed

    • Record seedling detachment after each pulse

    • Determine aerial tissue mass of each seedling

  • Data Analysis:

    • Calculate detachment risk relative to wild-type controls

    • Use statistical models to determine significance of adhesion differences

    • Present data as hazard ratios with confidence intervals

This methodology allows for quantitative comparison between wild-type and mutant lines, enabling detailed functional characterization of ABCG43's role in root-substrate interactions.

How can immunolocalization approaches be optimized for studying ABCG43 in plant tissues?

Optimizing immunolocalization for membrane-localized ABC transporters like ABCG43 requires:

  • Tissue Preparation:

    • Fix root tissues in 4% paraformaldehyde

    • Consider using a vacuum infiltration step to improve antibody penetration

    • For membrane proteins, include a mild detergent treatment to improve antigen accessibility

  • Antigen Retrieval:

    • Use citrate buffer (pH 6.0) heated treatment to expose epitopes

    • For membrane proteins like ABCG43, optimize detergent concentration in blocking and washing buffers

  • Blocking and Antibody Incubation:

    • Use BSA or normal serum in PBS with 0.1% Triton X-100

    • Incubate with primary antibody against ABCG43 overnight at 4°C

    • Use fluorophore-conjugated secondary antibodies for detection

  • Controls and Validation:

    • Include abcg43 mutant tissues as negative controls

    • Use known cellular markers to confirm subcellular localization

    • Compare patterns with GFP-tagged ABCG43 complementation lines if available

  • Imaging:

    • Employ confocal microscopy for precise localization

    • Consider super-resolution techniques for detailed membrane localization studies

This approach allows for visualization of ABCG43's distribution in different cell types and developmental stages, providing insights into its functional role.

What are the key considerations when screening for novel ABCG43 interaction partners?

When identifying proteins that interact with ABCG43:

  • Yeast Two-Hybrid Screening:

    • Design baits using hydrophilic domains of ABCG43

    • Screen against Arabidopsis cDNA libraries

    • Validate interactions using multiple reporter systems

  • Co-Immunoprecipitation:

    • Use anti-ABCG43 antibodies to pull down native protein complexes

    • Alternatively, express tagged versions of ABCG43 in planta

    • Analyze precipitated proteins by mass spectrometry

    • Validate with reciprocal co-IPs

  • Proximity Labeling Approaches:

    • Fuse ABCG43 with enzymes like BioID or TurboID

    • Express in Arabidopsis to biotinylate proximal proteins

    • Purify biotinylated proteins and identify by mass spectrometry

  • Split-GFP Complementation:

    • Fuse ABCG43 with one half of split GFP

    • Test candidate interactors fused to complementary GFP fragment

    • Visualize interactions in planta through reconstituted fluorescence

These approaches can reveal ABCG43's involvement in protein complexes that regulate root-substrate adhesion.

How can researchers quantify ABCG43 expression levels in different plant tissues and conditions?

To accurately quantify ABCG43 expression:

  • RT-qPCR Analysis:

    • Design specific primers spanning exon-exon junctions

    • Extract RNA from different tissues (roots, shoots, etc.)

    • Normalize expression to stable reference genes

    • Compare expression levels across developmental stages or treatments

  • Western Blotting:

    • Extract membrane proteins using appropriate buffers

    • Separate proteins using SDS-PAGE

    • Transfer to membrane and probe with anti-ABCG43 antibody

    • Quantify relative protein levels using densitometry

  • Reporter Gene Constructs:

    • Generate ABCG43 promoter::GUS or ABCG43 promoter::GFP fusions

    • Transform into Arabidopsis

    • Visualize expression patterns in different tissues

    • Quantify fluorescence intensity for relative expression levels

  • RNA-Seq Analysis:

    • Perform transcriptome analysis of different tissues or conditions

    • Identify differential expression of ABCG43

    • Correlate with expression of other genes to identify co-regulated networks

These complementary approaches provide comprehensive insights into ABCG43 expression patterns and regulation.

What approaches can be used to analyze the impact of ABCG43 mutations on plant phenotypes beyond root adhesion?

To characterize broader phenotypic impacts of ABCG43 mutation:

  • Morphological Analysis:

    • Measure root hair length and density using microscopy and image analysis software

    • Analyze at least 30 root hairs per plant across 8-10 individual plants

    • Quantify growth parameters (primary root length, lateral root number, etc.)

    • Assess aerial tissue development under different conditions

  • Stress Response Testing:

    • Subject plants to various abiotic stressors (drought, salt, heavy metals)

    • Measure survival rates, growth parameters, and physiological responses

    • Compare stress hormone levels between wild-type and mutant plants

  • Metabolomic Analysis:

    • Collect root exudates from wild-type and abcg43 mutants

    • Perform LC-MS or GC-MS to identify differential metabolite profiles

    • Focus on compounds potentially involved in root-substrate interactions

  • Transcriptomic Profiling:

    • Perform RNA-seq on root tissues from wild-type and mutant plants

    • Identify differentially expressed genes to reveal affected pathways

    • Use Gene Ontology enrichment to identify biological processes impacted

This multi-faceted approach can reveal ABCG43's broader roles beyond the initially identified root adhesion phenotype.

What are the common technical challenges in developing specific antibodies against plant ABC transporters like ABCG43?

Researchers face several challenges when developing antibodies against plant ABC transporters:

  • Membrane Protein Antigenicity Issues:

    • ABC transporters contain multiple transmembrane domains that are poorly immunogenic

    • Solution: Target hydrophilic loops or N/C-terminal regions for antibody production

  • Cross-Reactivity Concerns:

    • The ABC transporter family has many members with similar sequences

    • Solution: Perform detailed sequence alignments to identify unique epitopes specific to ABCG43

  • Expression System Limitations:

    • Plant membrane proteins often express poorly in heterologous systems

    • Solution: Consider using plant seed-based expression systems which have shown success for recombinant antibody production

  • Endoplasmic Reticulum Stress:

    • High-level expression can trigger unfolded protein response in expression systems

    • Solution: Monitor and optimize expression levels; seed-specific expression systems have shown tolerance to ER stress during antibody production

  • Validation Challenges:

    • Limited availability of purified native protein for validation

    • Solution: Use abcg43 mutant tissues as negative controls and heterologous expression systems for positive controls

Addressing these challenges requires careful experimental design and validation strategies.

How can the centrifuge-based assay be optimized and adapted for different experimental conditions?

The centrifuge-based root adhesion assay can be optimized in several ways:

  • Media Composition Variations:

    • Modify nutrient concentrations to study effects on adhesion

    • Add specific compounds to test hypotheses about ABCG43 function

    • Use different gel strengths to mimic various soil conditions

  • Environmental Condition Modifications:

    • Test different light intensities and photoperiods

    • Vary temperature and humidity conditions

    • Apply hormones or stress conditions prior to centrifugation

  • Methodological Refinements:

    • Adjust centrifugation speed increments for more precise measurements

    • Modify spin duration to capture time-dependent adhesion properties

    • Standardize seedling size and age for more consistent results

  • Data Analysis Enhancements:

    • Implement automated image analysis for detachment detection

    • Develop mathematical models that account for root architecture variables

    • Correlate adhesion measurements with molecular or cellular parameters

  • Equipment Adaptations:

    • Design custom plate holders for different growth container formats

    • Implement real-time imaging during centrifugation when possible

    • Create scaled-up versions for larger plant species

These optimizations expand the utility of the centrifuge assay beyond its original application.

What statistical approaches are most appropriate for analyzing ABCG43 functional data?

When analyzing data from ABCG43 functional studies, consider these statistical approaches:

  • Survival Analysis for Adhesion Data:

    • Use Cox proportional hazards models for centrifuge assay data

    • This approach accounts for right-censored data (plants remaining attached at maximum speed)

    • Present results as hazard ratios with confidence intervals

    • Example from literature: abcg43-1 showed detachment risk 0.245 times that of wild-type (95% CI: 0.172-0.348)

  • Mixed Effects Models for Growth Data:

    • Account for random effects of experimental batches

    • Include fixed effects of genotype, treatment, and their interactions

    • Use appropriate post-hoc tests with multiple comparison corrections

  • Multivariate Analysis for Complex Phenotypes:

    • Implement principal component analysis for datasets with multiple phenotypic measurements

    • Use MANOVA when comparing multiple dependent variables simultaneously

    • Consider partial least squares discriminant analysis for metabolomic datasets

  • Power Analysis for Experiment Design:

    • Determine appropriate sample sizes based on expected effect sizes

    • For root hair measurements, analyze at least 30 root hairs per plant across 8-10 individual plants

    • For adhesion assays, use sample sizes of ≥70 individuals for reliable detection of differences

  • Visualization Techniques:

    • Use Kaplan-Meier curves for presenting centrifuge assay results

    • Employ box plots with individual data points for transparent representation of variability

    • Consider heatmaps for presenting comprehensive datasets across conditions or genotypes

How might CRISPR/Cas9 genome editing be applied to study ABCG43 function?

CRISPR/Cas9 technology offers powerful approaches for studying ABCG43:

  • Precise Gene Knockout:

    • Design guide RNAs targeting critical exons of ABCG43

    • Generate complete knockouts to compare with T-DNA insertion alleles

    • Create tissue-specific knockouts using promoter-driven Cas9 expression

  • Domain-Specific Modifications:

    • Introduce point mutations in ATP-binding domains to study transporter activity

    • Modify specific amino acids in substrate-binding regions

    • Create truncation mutations to study domain function

  • Reporter Gene Knock-ins:

    • Insert fluorescent protein tags in-frame with ABCG43

    • Create endogenous promoter-reporter fusions at the native locus

    • Design split-reporter systems for protein interaction studies

  • Multiplexed Editing:

    • Target ABCG43 along with potential interaction partners

    • Create higher-order mutants to study genetic redundancy

    • Modify multiple members of the ABCG subfamily simultaneously

  • Base Editing Applications:

    • Introduce specific amino acid changes without double-strand breaks

    • Create allelic series to study structure-function relationships

    • Modify regulatory elements to study expression control

These approaches allow for more precise genetic manipulation than traditional T-DNA insertion methods.

What emerging technologies might advance our understanding of ABCG43 transport activity?

Several cutting-edge technologies show promise for characterizing ABCG43 function:

  • Single-Cell Omics:

    • Apply single-cell RNA-seq to map ABCG43 expression at cellular resolution

    • Use single-cell proteomics to quantify ABCG43 protein levels across cell types

    • Implement spatial transcriptomics to correlate expression with tissue locations

  • Advanced Imaging Techniques:

    • Apply FRET-based biosensors to monitor substrate transport in real-time

    • Use super-resolution microscopy to visualize ABCG43 localization in membrane microdomains

    • Implement light-sheet microscopy for whole-root imaging of ABCG43-GFP dynamics

  • Membrane Transport Assays:

    • Develop fluorescent substrate analogs to track ABCG43 transport activity

    • Use electrophysiological approaches in heterologous expression systems

    • Implement vesicle-based transport assays with purified ABCG43 protein

  • Structural Biology Approaches:

    • Apply cryo-EM to determine ABCG43 structure in different conformational states

    • Use hydrogen-deuterium exchange mass spectrometry to map substrate binding sites

    • Implement molecular dynamics simulations to model transport mechanisms

  • Synthetic Biology Strategies:

    • Create chimeric transporters to identify functional domains

    • Develop optogenetic tools to control ABCG43 activity with light

    • Design synthetic circuits to modulate ABCG43 expression in specific contexts

These technologies will provide deeper insights into the biochemical and cellular functions of ABCG43.

How might research on ABCG43 contribute to improving crop resilience to environmental stresses?

Understanding ABCG43's function in root-substrate interactions has implications for crop improvement:

  • Translational Research Opportunities:

    • Identify homologs of ABCG43 in crop species such as rice, wheat, and maize

    • Determine if similar root adhesion phenotypes exist in crop ABCG43 orthologs

    • Assess whether modifying ABCG43 activity improves root anchorage in agricultural settings

  • Drought Resistance Applications:

    • Investigate whether ABCG43-mediated changes in root adhesion affect water uptake efficiency

    • Test if enhanced root-substrate contact improves drought tolerance

    • Develop crop varieties with optimized ABCG43 expression for water-limited environments

  • Nutrient Acquisition Enhancement:

    • Study how ABCG43-related root adhesion affects nutrient uptake from soil

    • Investigate potential roles in mycorrhizal associations and nutrient exchange

    • Target ABCG43 modification to improve phosphorus acquisition in low-input systems

  • Soil Erosion Mitigation:

    • Assess if ABCG43 variants with enhanced adhesion can improve soil stability

    • Test potential for reduced erosion in agricultural systems

    • Develop cover crops with enhanced root adhesion for conservation agriculture

  • Rhizosphere Engineering:

    • Investigate how ABCG43 affects root exudate composition and microbial interactions

    • Determine if modified ABCG43 activity can enhance beneficial microbial associations

    • Develop crops with optimized rhizosphere communities for sustainable agriculture

These applications highlight the potential broader impacts of fundamental research on ABCG43 function.

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