At3g05155 Antibody

What is the AT3G05155 protein and why is it significant for plant research?

AT3G05155 encodes a Major Facilitator Superfamily (MFS) protein that functions as a transmembrane transporter facilitating the movement of small solutes across cellular membranes via electrochemical gradients. This protein belongs to one of the largest transporter families in plants, with critical roles in nutrient uptake, metabolite export, and stress response signaling. Understanding its function provides valuable insights into plant nutrient homeostasis, development, and environmental adaptation mechanisms. Though its specific substrate remains uncharacterized, research suggests it likely transports sugars or related metabolites, positioning it as a potentially important component in plant carbon allocation and stress physiology research.

How does the AT3G05155 antibody compare to antibodies against other MFS transporters?

The AT3G05155 antibody targets a specific epitope of this MFS transporter protein (UniProt ID: Q7XA64). Unlike antibodies against better-characterized plant transporters such as SUC/SUT sucrose transporters or hexose transporters, the At3g05155 antibody targets a relatively understudied MFS member. When designing experimental controls, researchers should consider using antibodies against structurally similar but functionally distinct MFS transporters. This approach allows discrimination between specific and non-specific binding patterns, particularly important in immunolocalization studies where cross-reactivity can confound results. While comparative data is limited in the literature, preliminary findings suggest standard validation protocols including western blotting with recombinant protein controls can effectively establish antibody specificity.

What are the optimal fixation methods for immunolocalization studies using the At3g05155 antibody?

For immunolocalization of AT3G05155 protein in plant tissues, researchers should consider both tissue preservation quality and epitope accessibility. Based on protocols established for similar MFS transporters in Arabidopsis, a sequential fixation approach is recommended:

• Primary fixation with 4% paraformaldehyde in PBS (pH 7.4) for 2-4 hours at 4°C

• Post-fixation in 0.1% glutaraldehyde for 30 minutes (if enhanced structural preservation is required)

• Careful dehydration series using gradual ethanol concentrations (30%, 50%, 70%, 90%, 100%)

• Embedding in a medium like LR White resin for sectioning

For whole-mount immunolocalization, mild permeabilization with 0.1% Triton X-100 after fixation improves antibody penetration while preserving membrane structures where the transporter is located. Comparing different fixation protocols is advisable during assay optimization, as overfixation can mask epitopes while underfixation compromises tissue integrity . Research on related membrane transporters indicates that phosphate-buffered fixatives generally provide superior results compared to Tris-buffered alternatives when studying membrane-localized proteins.

How can I validate the specificity of the At3g05155 antibody in my experimental system?

Validating At3g05155 antibody specificity requires a multi-faceted approach:

• Genetic controls: The most stringent validation employs knockout/knockdown lines (T-DNA insertion mutants) of AT3G05155. The absence or significant reduction of signal in these lines compared to wild-type provides strong evidence for antibody specificity .

• Western blot analysis: Perform western blots with total protein extracts from wild-type plants and at3g05155 mutants. The antibody should detect a band at the predicted molecular weight (~55-60 kDa) in wild-type samples that is absent or significantly reduced in mutant samples .

• Peptide competition assay: Pre-incubate the antibody with the synthetic peptide used for immunization. This should abolish specific binding in subsequent immunoassays.

• Recombinant protein controls: Express the AT3G05155 protein (or its immunogenic domain) in a heterologous system and verify detection by the antibody.

• Cross-reactivity assessment: Test the antibody against closely related MFS family members to ensure it doesn't recognize similar epitopes in related proteins.

Documentation of these validation steps is essential for publishing studies that rely on this antibody, as membrane protein antibodies often require extensive validation due to potential cross-reactivity with structurally similar transporters .

What are the optimal protein extraction conditions for detecting AT3G05155 in western blots?

Efficient extraction of AT3G05155 membrane protein requires specialized buffers and protocols:

• Extraction buffer composition:

  • Base buffer: 50 mM Tris-HCl (pH 7.5), 150 mM NaCl

  • Detergents: 1% Triton X-100 or 0.5% sodium deoxycholate

  • Protease inhibitors: Complete Protease Inhibitor Cocktail

  • Reducing agents: 5 mM DTT or 10 mM β-mercaptoethanol

  • Membrane stabilizers: 10% glycerol

• Extraction protocol:

  • Homogenize plant tissue in cold extraction buffer (4:1 buffer:tissue ratio)

  • Incubate homogenate at 4°C for 30-60 minutes with gentle rotation

  • Centrifuge at 10,000g for 15 minutes to remove cell debris

  • Ultracentrifuge supernatant at 100,000g for 1 hour to obtain membrane fraction

  • Resuspend membrane pellet in SDS-PAGE sample buffer containing 2% SDS

• Sample preparation:

  • Avoid boiling samples (heat to 37°C for 30 minutes instead)

  • Load 20-50 μg total membrane protein per lane

  • Use 10-12% acrylamide gels for optimal resolution

This protocol minimizes protein aggregation and degradation while maximizing extraction efficiency from membrane fractions. For challenging tissues like mature leaves with high phenolic content, adding 1-2% polyvinylpolypyrrolidone (PVPP) to the extraction buffer can improve results by absorbing interfering compounds .

What controls should be included when performing RT-PCR to analyze AT3G05155 expression?

When analyzing AT3G05155 expression via RT-PCR, include these essential controls:

• Endogenous reference genes:

  • Primary reference: ACT2 (Actin 2) or UBQ10 (Ubiquitin 10)

  • Secondary reference: EF1α (Elongation Factor 1α) or GAPDH

  • Tissue-specific reference: APT1 (Adenine Phosphoribosyltransferase 1) for roots

• Negative controls:

  • No-template control (NTC)

  • Reverse transcriptase-minus control (RT-)

  • Genomic DNA control (to detect genomic contamination)

• Positive controls:

  • Tissues known to express AT3G05155 (vascular tissues show consistent expression)

  • Plasmid containing AT3G05155 cDNA (for standard curve generation)

• Expression validation controls:

  • Alternative primer sets targeting different regions of AT3G05155

  • Analysis of splice variants using primers spanning exon junctions

The recommended primer design parameters include:

• Amplicon size: 80-150 bp for qRT-PCR, 400-600 bp for standard RT-PCR

• Primer melting temperature: 58-62°C

• GC content: 40-60%

• Exon-spanning design to avoid genomic DNA amplification

In stress response studies, include well-characterized stress marker genes as positive controls for the experimental treatment, such as RD29A for drought/salt stress or PR1 for pathogen response .

How does AT3G05155 expression change in response to biotic and abiotic stresses?

The expression pattern of AT3G05155 shows notable modulation under various stress conditions:

The upregulation during pathogen infection suggests a potential role in plant defense responses, possibly through altered carbohydrate partitioning or signaling molecule transport . The involvement in powdery mildew response is particularly notable, as infection by these biotrophic pathogens creates localized nutrient sinks that may require increased transport activity. Similar MFS transporters are known to participate in the redistribution of photosynthates during pathogen infection, potentially supporting defense compound synthesis or altered source-sink relationships .

For biotic stress studies, monitoring AT3G05155 expression alongside established pathogen-responsive genes like PR1 (pathogenesis-related 1) provides context for the temporal dynamics of the response. The moderate upregulation during drought and salt stress further suggests a possible role in osmotic adjustment or compatible solute transport, mechanisms known to involve MFS transporters in abiotic stress adaptation .

What protein-protein interactions have been identified for the AT3G05155 protein?

Limited direct interaction data exists for AT3G05155, but analyses of related MFS transporters suggest potential interaction partners:

While direct evidence for AT3G05155 interactions requires further investigation, analysis of co-expression networks identifies associations with other membrane transporters involved in nutrient trafficking. The potential connection to GRXS17 is particularly intriguing, as glutaredoxins like GRXS17 participate in iron homeostasis and Fe-S cluster assembly pathways, which could influence transporter activity through post-translational modifications .

To investigate these interactions experimentally, researchers should consider:

• Split-ubiquitin yeast two-hybrid assays (optimized for membrane proteins)

• Bimolecular fluorescence complementation (BiFC) in Arabidopsis protoplasts

• Co-immunoprecipitation with AT3G05155 antibody followed by mass spectrometry

• Proximity-dependent biotin identification (BioID) using AT3G05155 as bait

These approaches could reveal whether AT3G05155 functions independently or as part of larger transport complexes that coordinate nutrient movement across cellular membranes .

How can I use the At3g05155 antibody to investigate subcellular trafficking dynamics?

Investigating AT3G05155 trafficking dynamics requires techniques that capture both spatial and temporal aspects of protein localization:

• Pulse-chase immunolocalization:

  • Transform plants with an inducible AT3G05155-tag fusion construct

  • Induce expression briefly (pulse)

  • Block further synthesis (chase)

  • Fix samples at different timepoints

  • Immunolocalize using the At3g05155 antibody

  • Quantify signal intensity at different cellular compartments

• Co-localization with endomembrane markers:

  • Perform double immunofluorescence with the At3g05155 antibody and antibodies against:

    • TGN markers (SYP61, VHA-a1)

    • Golgi markers (α-mannosidase II)

    • Plasma membrane markers (PIP2;1)

    • Endocytic markers (RABF2b/ARA7)

  • Calculate Pearson's correlation coefficients between signals

  • Monitor changes in co-localization patterns under different conditions

• Superresolution microscopy approaches:

  • Structured illumination microscopy (SIM) offers 2x resolution improvement

  • Stimulated emission depletion (STED) microscopy for 50nm resolution

  • Single-molecule localization microscopy for trafficking pathway mapping

• Vesicle isolation and immunoblotting:

  • Isolate different membrane fractions via density gradient centrifugation

  • Probe fractions with the At3g05155 antibody

  • Quantify protein distribution across fractions

For dynamic trafficking studies, researchers should consider stress conditions that may trigger relocalization, such as pathogen exposure or nutrient deprivation. Inhibitor treatments (Brefeldin A, wortmannin, etc.) can help dissect the trafficking pathways involved in AT3G05155 delivery to its target membrane .

What are common troubleshooting strategies for weak signals when using the At3g05155 antibody?

When encountering weak signal issues with the At3g05155 antibody, consider the following systematic troubleshooting approach:

• Sample preparation issues:

  • Increase protein extraction efficiency by using stronger detergents (1% SDS)

  • Optimize membrane protein solubilization with different detergent combinations

  • Minimize protein degradation with fresh protease inhibitors

  • Concentrate membrane fractions through ultracentrifugation

• Antibody-specific optimizations:

  • Test different antibody dilutions (1:500 to 1:5000 range)

  • Extend primary antibody incubation time (overnight at 4°C)

  • Try different blocking agents (5% milk, 3% BSA, commercial blockers)

  • Use signal enhancement systems (biotin-streptavidin, tyramide)

• Detection system improvements:

  • Switch to more sensitive detection substrates (femto-level ECL)

  • Try alternative secondary antibodies

  • Use polymer-based detection systems for immunohistochemistry

  • Consider fluorescent secondary antibodies with longer exposure times

• Tissue-specific considerations:

  • For recalcitrant tissues, modify fixation protocols (reduce time/concentration)

  • For tissues with high autofluorescence, use Sudan Black B treatment

  • For tissues with high phenolic content, add PVPP to extraction buffers

If weak signals persist despite optimization, consider protein abundance issues. AT3G05155 may be expressed at low levels under normal conditions, necessitating protein concentration steps or expression in conditions known to upregulate the gene, such as during pathogen infection or specific stress conditions .

How can I correctly interpret AT3G05155 western blot results when multiple bands appear?

Multiple bands in AT3G05155 western blots require careful interpretation:

• Expected pattern interpretation:

  • Primary band: ~55-60 kDa (predicted molecular weight)

  • Higher bands (90-120 kDa): Potential dimers/multimers (common in membrane transporters)

  • Lower bands (30-40 kDa): Possible degradation products or proteolytic processing

• Validation approaches for multiple bands:

  • Compare band patterns between wild-type and at3g05155 mutant tissues

  • Perform peptide competition assays to identify specific bands

  • Use different protein extraction methods to determine if bands are artifacts

  • Test different reducing conditions to assess multimer formation

• Post-translational modification assessment:

  • Treat samples with:

    • Phosphatase (to remove phosphorylation)

    • PNGase F (to remove N-linked glycosylation)

    • Endoglycosidase H (to remove high-mannose glycans)

  • Observe resulting band pattern shifts

• Analytical approaches:

  • Perform 2D gel electrophoresis to separate by both pI and molecular weight

  • Use mass spectrometry to confirm protein identity in excised bands

  • Analyze multiple tissues to determine if band patterns are tissue-specific

A reference band pattern table for different sample preparation conditions:

When publishing results, clearly document which band is being quantified and provide evidence for its specificity to AT3G05155 .

How can I design experiments to study the functional relationship between AT3G05155 and GRXS17 in Fe-S protein metabolism?

The potential functional relationship between AT3G05155 and GRXS17 in Fe-S protein metabolism can be investigated through these experimental approaches:

• Genetic interaction studies:

  • Generate at3g05155 grxs17 double mutants

  • Compare phenotypes of single and double mutants

  • Assess epistatic relationships through severity of growth, development, and stress response phenotypes

  • Perform complementation studies with wild-type and mutated versions of each gene

• Biochemical interaction analysis:

  • Perform split-ubiquitin yeast two-hybrid assays optimized for membrane proteins

  • Conduct co-immunoprecipitation experiments using At3g05155 and GRXS17 antibodies

  • Test direct interaction using recombinant proteins in vitro

  • Employ bimolecular fluorescence complementation to visualize interactions in vivo

• Functional metabolic studies:

  • Measure activity of Fe-S enzymes (e.g., aconitase, xanthine dehydrogenase) in single and double mutants

  • Quantify iron content and distribution using Perls' staining and ICP-MS

  • Analyze expression of iron homeostasis genes in response to iron availability

  • Examine ROS production and oxidative stress markers

• Transporter activity assessment:

  • Measure substrate transport in heterologous expression systems (yeast, oocytes)

  • Compare transport activity with and without functional GRXS17

  • Test transport under different redox conditions

  • Analyze post-translational modifications that might be affected by GRXS17

A systematic experimental matrix for investigating this relationship:

This comprehensive approach would provide multiple lines of evidence regarding whether AT3G05155 functions within the Fe-S cluster assembly/delivery pathway potentially coordinated by GRXS17. The research would contribute to understanding how membrane transport processes intersect with cellular iron homeostasis and Fe-S protein maturation pathways .

How might At3g05155 antibodies be used to investigate the connection between sugar transport and plant immunity?

The At3g05155 antibody offers valuable opportunities to explore the intersection of sugar transport and plant immunity:

• Spatial-temporal profiling during pathogen infection:

  • Use immunolocalization to track AT3G05155 protein redistribution during:

    • Early pathogen recognition (0-12 hours post-infection)

    • Effector-triggered immunity responses (12-24 hours)

    • Systemic acquired resistance development (24-72 hours)

  • Compare compatible vs. incompatible interactions to identify defense-specific patterns

  • Correlate localization changes with sugar concentration dynamics

• Infection site-specific analysis:

  • Apply laser-capture microdissection coupled with immunoblotting to analyze:

    • Infected cells

    • Adjacent non-infected cells

    • Distant systemic tissues

  • Quantify AT3G05155 protein abundance in these distinct zones

  • Compare with known defense-related transporters

• Co-regulation with immune signaling components:

  • Perform co-immunoprecipitation during infection progression to identify:

    • Immune receptor associations

    • Interactions with known defense signaling hubs

    • Post-translational modifications specific to defense activation

  • Test if immune signaling molecules directly regulate transporter activity

• Functional studies in immunity contexts:

  • Compare defense responses in wild-type vs. at3g05155 mutants:

    • Pathogen growth restriction capability

    • Callose deposition at infection sites

    • ROS burst intensity and duration

    • PR gene expression profiles

  • Complement with transport-deficient protein variants

Current research on powdery mildew interactions shows a 2.4-fold increase in AT3G05155 expression during infection, suggesting its involvement in pathogen-induced metabolic reprogramming . This upregulation may reflect the plant's strategy to restrict nutrient availability to the pathogen or to redirect resources toward defense compound synthesis. The antibody enables protein-level verification of these transcriptional changes and allows investigation of potential post-translational regulation mechanisms that may fine-tune transporter activity during immune responses .

What techniques can be used to investigate AT3G05155 protein turnover and regulation?

Investigating AT3G05155 protein turnover and regulation requires specialized techniques to capture the dynamic nature of membrane protein homeostasis:

• Protein stability and half-life determination:

  • Cycloheximide chase assays: Treat plants with protein synthesis inhibitor and monitor AT3G05155 abundance over time

  • Pulse-chase with inducible expression systems

  • Quantitative western blotting with the At3g05155 antibody at defined intervals

  • Mathematical modeling to calculate degradation rates

• Post-translational modification mapping:

  • Immunoprecipitate AT3G05155 using the antibody

  • Analyze by mass spectrometry to identify:

    • Phosphorylation sites

    • Ubiquitination

    • S-nitrosylation

    • Other redox-based modifications

  • Compare modification patterns under different conditions

• Degradation pathway analysis:

  • Treat plants with specific inhibitors:

    • MG132 (proteasome inhibitor)

    • Concanamycin A (vacuolar degradation inhibitor)

    • E64d (cysteine protease inhibitor)

  • Monitor AT3G05155 abundance changes by immunoblotting

  • Use fluorescently tagged versions to track subcellular localization during degradation

• Regulatory element identification:

  • Map protein domains involved in trafficking/degradation using deletion variants

  • Identify sequence motifs mediating endocytosis or recycling

  • Test chimeric proteins with domains from related stable/unstable transporters

A sample experimental workflow for AT3G05155 turnover analysis:

The findings from these experiments would reveal how AT3G05155 protein levels are regulated post-translationally, complementing transcriptional studies to provide a comprehensive understanding of transporter regulation under different physiological conditions .

How conserved is AT3G05155 across plant species and what insights does this provide for antibody cross-reactivity?

Evolutionary analysis of AT3G05155 reveals important patterns relevant to antibody applications across species:

• Conservation pattern across plant lineages:

  • Highly conserved in Brassicaceae (>85% amino acid identity)

  • Moderately conserved in other dicots (60-75% identity)

  • Less conserved in monocots (40-55% identity)

  • Limited conservation in non-vascular plants (<30% identity)

• Domain-specific conservation:

  • Transmembrane domains: Highest conservation (70-90% across angiosperms)

  • Central loop region: Moderate conservation (40-60%)

  • N and C termini: Lowest conservation (20-40%)

  • MFS signature motifs: Nearly invariant across all plant species

• Epitope conservation for antibody applications:

  • The At3g05155 antibody targets an epitope in the protein's C-terminal region

  • Sequence alignment reveals high conservation within Brassicaceae

  • Limited conservation in non-Brassicaceae species

• Cross-reactivity prediction by taxon:

The evolutionary conservation patterns suggest that the At3g05155 antibody would be most reliable for studies within Arabidopsis and closely related Brassicaceae. For research in other plant families, the antibody should be rigorously validated or species-specific alternatives should be developed. The highly conserved transmembrane domains could potentially serve as targets for developing broader-specificity antibodies, though this would require careful design to avoid cross-reactivity with other MFS family members.

This evolutionary context also provides insight into the functional importance of different protein regions. The high conservation of transmembrane domains suggests strong selective pressure on transport function, while the variable terminal regions may reflect species-specific regulatory adaptations.

How do current hypotheses about AT3G05155 function connect to broader plant metabolism?

Current hypotheses about AT3G05155 function intersect with several key metabolic pathways:

• Carbon partitioning and source-sink dynamics:

  • As a putative sugar transporter, AT3G05155 may influence carbohydrate distribution

  • Expression increases during powdery mildew infection suggest involvement in pathogen-induced sink formation

  • Potential role in coordinating photosynthate allocation between growth and defense

• Connections to iron homeostasis and Fe-S protein function:

  • Co-purification with GRXS17 suggests potential involvement in Fe-S cluster metabolism

  • May transport substrates required for Fe-S cluster assembly or distribution

  • Could function in signaling pathways connecting nutrient transport to iron status

• Stress response metabolic reprogramming:

  • Upregulation during specific stresses indicates involvement in metabolic adaptation

  • May transport compatible solutes or signaling molecules during stress responses

  • Could facilitate resource reallocation during acclimation processes

• Integration with plant development:

  • Expression patterns correlate with developmental transitions

  • Potential role in providing substrates for phase change-associated metabolic shifts

  • May coordinate with hormonal signaling networks regulating development

A conceptual framework connecting AT3G05155 to broader metabolic networks:

These hypotheses present several testable predictions for future research. For example, at3g05155 mutants might show altered metabolite profiles during stress responses, changed iron distribution, or modified susceptibility to biotrophic pathogens. The At3g05155 antibody would be valuable for testing these hypotheses by tracking protein abundance and localization under relevant conditions .

What emerging technologies might enhance At3g05155 antibody applications in plant research?

Several cutting-edge technologies could significantly expand the utility of At3g05155 antibodies:

• Super-resolution microscopy techniques:

  • Stochastic optical reconstruction microscopy (STORM) can achieve 20nm resolution

  • DNA-PAINT approaches for multiplexed protein detection

  • Expansion microscopy to physically enlarge samples

  • Applications: Nanoscale mapping of AT3G05155 distribution in membrane microdomains

• Single-cell proteomics integration:

  • Mass cytometry (CyTOF) with metal-conjugated At3g05155 antibodies

  • Microfluidic antibody-based sorting of protoplasts

  • Applications: Cell type-specific transporter abundance profiles across tissues

• Proximity labeling approaches:

  • BioID or TurboID fusion proteins to identify proximal interactors

  • APEX2-based spatial proteomics

  • Applications: Mapping the dynamic AT3G05155 protein interaction network

• Antibody engineering advancements:

  • Single-chain variable fragments for improved penetration

  • Split-antibody complementation assays

  • pH-sensitive fluorescent antibody conjugates

  • Applications: Real-time monitoring of trafficking in live cells

• Cryo-electron microscopy applications:

  • Antibody-based localization in cryo-preserved samples

  • Structural studies using antibody fragments

  • Applications: Correlative light-electron microscopy of transporter complexes

Each of these technologies addresses current limitations in studying AT3G05155 biology. For example, conventional confocal microscopy lacks the resolution to determine if AT3G05155 localizes to specific membrane microdomains, which could be resolved using super-resolution approaches. Similarly, single-cell techniques would reveal cell-to-cell variation in transporter abundance that is masked in whole-tissue analyses, potentially uncovering specialized cell types with high expression .

These advanced methods, when combined with genetic tools like CRISPR-engineered variants and environmental manipulations, promise to provide unprecedented insights into the dynamic regulation and function of this transporter in plant physiology.