At3g05165 Antibody

What is At3g05165 and why are antibodies against it important in plant research?

At3g05165 is a gene in Arabidopsis thaliana (Mouse-ear cress) that encodes a Sugar transporter ERD6-like 11, which belongs to the Major facilitator superfamily protein . This protein plays a crucial role in sugar transport mechanisms in plants, particularly under stress conditions.

Antibodies against At3g05165 are important research tools because they allow scientists to:

• Study protein expression levels in different tissues

• Examine protein localization within cells

• Investigate protein-protein interactions

• Monitor changes in protein expression under various environmental conditions

The antibodies provide a specific molecular probe for detecting this sugar transporter, which is essential for understanding plant carbohydrate metabolism and stress responses.

What types of At3g05165 antibodies are available for research?

Currently, the most common type of At3g05165 antibody available is a rabbit polyclonal antibody that targets Arabidopsis thaliana At3g05165 . These antibodies are typically:

Unlike monoclonal antibodies that recognize a single epitope, these polyclonal antibodies bind to multiple epitopes on the At3g05165 protein, making them useful for detection even when some epitopes might be masked or altered during experimental procedures.

How specific are At3g05165 antibodies for their target protein?

The specificity of At3g05165 antibodies is determined through several validation methods:

• Western blotting against wild-type and knockout plant extracts

• Immunoprecipitation followed by mass spectrometry analysis

• Competitive binding assays with purified recombinant protein

• Cross-reactivity testing against closely related proteins

Research indicates that while these antibodies are highly specific for At3g05165, there may be minimal cross-reactivity with other members of the ERD6-like sugar transporter family due to sequence homology, particularly with At3g05160 (Sugar transporter ERD6-like 10) and At3g05155 (Sugar transporter ERD6-like 9) . This is an important consideration when designing experiments, and proper controls should be implemented.

What is the optimal protocol for using At3g05165 antibodies in Western blotting?

For optimal Western blot results with At3g05165 antibodies, follow this validated protocol:

Sample Preparation:

• Extract total protein from plant tissue using a buffer containing 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, and protease inhibitors

• Quantify protein concentration using Bradford assay

• Denature 20-40 μg of protein in Laemmli buffer at 70°C for 10 minutes (avoid higher temperatures which may cause aggregation of membrane proteins)

Electrophoresis and Transfer:

• Separate proteins on 10-12% SDS-PAGE gel

• Transfer to PVDF membrane (preferred over nitrocellulose for membrane proteins)

• Confirm transfer efficiency with reversible Ponceau S staining

Immunodetection:

• Block membrane with 5% non-fat milk in TBST for 1 hour at room temperature

• Incubate with At3g05165 antibody (1:1000 dilution) overnight at 4°C

• Wash 4× with TBST, 5 minutes each

• Incubate with HRP-conjugated secondary antibody (1:5000) for 1 hour

• Wash 4× with TBST, 5 minutes each

• Develop using ECL detection reagent

The expected molecular weight of At3g05165 protein is approximately 55-60 kDa, though post-translational modifications may result in slight variations in migration patterns .

How should At3g05165 antibodies be used in immunolocalization studies?

For successful immunolocalization of At3g05165 in plant tissues:

Tissue Fixation and Embedding:

• Fix fresh tissue in 4% paraformaldehyde in PBS (pH 7.4) for 4 hours

• Dehydrate through an ethanol series (30%, 50%, 70%, 90%, 100%)

• Infiltrate and embed in either paraffin for light microscopy or LR White resin for electron microscopy

Immunolabeling Protocol:

• For paraffin sections: Deparaffinize and rehydrate, then perform antigen retrieval using 10 mM sodium citrate buffer (pH 6.0) at 95°C for 10 minutes

• Block non-specific binding with 2% BSA in PBS for 1 hour

• Incubate with At3g05165 antibody (1:100-1:200) overnight at 4°C

• Wash 3× with PBS, 10 minutes each

• Incubate with fluorophore-conjugated secondary antibody for 2 hours at room temperature

• Counterstain nuclei with DAPI (1 μg/ml)

• Mount in anti-fade medium

Controls: Always include negative controls (primary antibody omitted) and, if possible, tissue from At3g05165 knockout plants to confirm specificity of the signal .

What are the recommended methods for using At3g05165 antibodies in ELISA applications?

For quantitative detection of At3g05165 protein using ELISA:

Indirect ELISA Protocol:

• Coat 96-well plates with plant extract diluted in carbonate-bicarbonate buffer (pH 9.6) overnight at 4°C

• Block with 3% BSA in PBS for 2 hours at room temperature

• Add At3g05165 antibody diluted 1:500-1:2000 in blocking buffer and incubate for 2 hours

• Wash 4× with PBST

• Add HRP-conjugated secondary antibody and incubate for 1 hour

• Wash 4× with PBST

• Develop with TMB substrate and stop reaction with 2M H₂SO₄

• Read absorbance at 450 nm

Sandwich ELISA (for higher specificity):

• Coat plates with a capture antibody (e.g., a different At3g05165 antibody recognizing a distinct epitope)

• Block and add sample

• Detect with the primary At3g05165 antibody followed by enzyme-conjugated secondary antibody

Sandwich ELISA typically provides increased specificity but requires two antibodies recognizing different epitopes on the At3g05165 protein .

How can At3g05165 antibodies be used to study sugar transport mechanisms in plants under stress conditions?

At3g05165 encodes an ERD6-like sugar transporter that is often upregulated during stress conditions. To study its role:

Stress Response Analysis Protocol:

• Subject plants to specific stresses (drought, salt, cold) for various durations

• Collect tissue samples at defined timepoints

• Perform Western blot analysis using At3g05165 antibodies to track protein expression changes

• Complement with RT-qPCR to correlate transcript and protein levels

• Use immunolocalization to determine if cellular distribution changes under stress

Functional Analysis:

• Compare wild-type and At3g05165 knockout plants using physiological parameters

• Measure sugar content in different cellular compartments using non-aqueous fractionation

• Use At3g05165 antibodies to immunoprecipitate the protein and identify interaction partners that may change under stress conditions

• Perform transport assays with membrane vesicles and confirm transporter presence using the antibody

This integrative approach can reveal how At3g05165 contributes to stress adaptation through sugar redistribution mechanisms .

How can co-immunoprecipitation with At3g05165 antibodies be optimized for identifying protein interaction partners?

To maximize success in co-immunoprecipitation (Co-IP) experiments:

Optimized Co-IP Protocol:

• Harvest 5-10g of plant tissue and grind in liquid nitrogen

• Extract proteins in buffer containing 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM EDTA, 10% glycerol, 0.1-0.5% NP-40, and protease inhibitors

• Clear lysate by centrifugation at 20,000×g for 20 minutes

• Pre-clear with Protein A/G beads for 1 hour at 4°C

• Incubate 1-2 mg protein with 2-5 μg At3g05165 antibody overnight at 4°C with gentle rotation

• Add Protein A/G beads and incubate for 3 hours at 4°C

• Wash beads 5× with extraction buffer containing reduced detergent (0.05% NP-40)

• Elute proteins with 2× Laemmli buffer at 70°C for 10 minutes

• Analyze by SDS-PAGE followed by silver staining or Western blotting

Critical Considerations:

• Use chemical crosslinking (e.g., DSP or formaldehyde) to stabilize transient interactions

• Test different detergent types and concentrations to maintain membrane protein solubility while preserving interactions

• Include negative controls using pre-immune serum or IgG from the same species

• Validate results using reciprocal Co-IP with antibodies against suspected interaction partners

This approach has successfully identified protein complexes involving membrane transporters in Arabidopsis .

How do post-translational modifications affect At3g05165 antibody recognition, and how can these modifications be studied?

Post-translational modifications (PTMs) can significantly impact antibody recognition of At3g05165:

Common PTMs Affecting Antibody Recognition:

• Phosphorylation - often occurs on serine/threonine residues in transporters

• Glycosylation - particularly relevant for plasma membrane proteins

• Ubiquitination - can signal protein degradation or trafficking

• SUMOylation - may regulate protein activity and localization

Methods to Study PTMs:

• Western Blot Analysis:

  • Use phospho-specific antibodies alongside generic At3g05165 antibodies

  • Treat samples with phosphatase, glycosidase, or deubiquitinating enzymes before Western blotting

  • Compare migration patterns before and after treatment

• Mass Spectrometry Approach:

  • Immunoprecipitate At3g05165 using the antibody

  • Digest with trypsin and analyze by LC-MS/MS

  • Search for modified peptides using appropriate software

• 2D Gel Electrophoresis:

  • Separate proteins by isoelectric point and molecular weight

  • Detect At3g05165 with the antibody

  • Multiple spots may indicate different PTM states

Understanding these modifications can provide insight into how At3g05165 activity is regulated in response to environmental cues or developmental stages .

What are the challenges in using At3g05165 antibodies for evolutionary studies across plant species?

Cross-species application of At3g05165 antibodies faces several challenges:

Evolutionary Considerations Table:

Recommended Approach for Cross-Species Studies:

• Perform sequence alignment of At3g05165 homologs across target species

• Identify conserved regions as potential epitopes

• Test antibody reactivity on recombinant proteins from different species

• Validate antibody specificity using overexpression or knockout lines when available

• Consider raising new antibodies against synthetic peptides representing conserved regions

Cross-species studies with At3g05165 antibodies have successfully demonstrated evolutionary conservation of sugar transport mechanisms across various plant families, though sensitivity may vary significantly .

What are the most common causes of false positives or weak signals when using At3g05165 antibodies, and how can they be addressed?

Common Issues and Solutions:

Verification Strategy:

• Perform peptide competition assay by pre-incubating antibody with immunizing peptide

• Test antibody on recombinant At3g05165 protein as positive control

• Compare antibody performance in wild-type vs. At3g05165 knockout/knockdown plants

• Use alternative detection methods (e.g., mass spectrometry) to confirm findings

Optimizing sample preparation is particularly critical for membrane proteins like At3g05165, as improper extraction can significantly reduce signal intensity .

How can At3g05165 antibodies be used in conjunction with CRISPR/Cas9 gene editing to validate gene function studies?

CRISPR/Cas9 editing combined with antibody detection provides powerful validation of gene function:

Integrated Validation Protocol:

• Design CRISPR/Cas9 constructs targeting At3g05165 at multiple sites

• Generate edited plants and confirm mutations by sequencing

• Extract proteins from wild-type and mutant plants

• Perform Western blotting with At3g05165 antibody to confirm protein absence/truncation

• Conduct phenotypic analysis correlating with protein expression data

• Use antibody for immunolocalization to confirm cellular absence in mutants

• For complementation studies, use the antibody to verify expression of the introduced transgene

Advanced Applications:

• Create epitope-tagged versions of At3g05165 using CRISPR-mediated homology-directed repair

• Compare detection between endogenous protein (using At3g05165 antibody) and tagged protein

• Use CRISPR to introduce specific mutations and assess effects on protein stability and localization using the antibody

This approach can distinguish between effects caused by protein absence versus mislocalization or altered function .

What approaches can improve the sensitivity of At3g05165 protein detection in samples with low abundance?

When dealing with low-abundance At3g05165 protein:

Signal Enhancement Strategies:

• Sample Enrichment:

  • Fractionate cells and focus on membrane fractions

  • Use affinity purification with lectins (for glycosylated forms)

  • Perform immunoprecipitation before Western blotting

• Detection Enhancement:

  • Use high-sensitivity ECL substrates (femtogram detection limits)

  • Apply tyramide signal amplification for immunohistochemistry

  • Implement biotin-streptavidin amplification systems

  • Consider quantum dot-conjugated secondary antibodies

• Instrumentation Optimization:

  • Use cooled CCD cameras for weak chemiluminescence

  • Apply spectral unmixing for immunofluorescence

  • Utilize photomultiplier tube detection for enhanced sensitivity

Comparative Sensitivity Table:

These approaches have successfully detected low-abundance membrane transporters in specialized plant cells where expression is typically limited .

How can At3g05165 antibodies contribute to understanding drought response mechanisms in crops?

At3g05165 encodes a sugar transporter involved in stress response pathways. Antibodies against this protein can elucidate drought response mechanisms:

Research Applications in Drought Studies:

• Temporal Expression Profiling:

  • Track At3g05165 protein levels throughout drought stress progression

  • Compare expression patterns between drought-sensitive and drought-resistant varieties

  • Correlate protein accumulation with physiological parameters

• Spatial Distribution Analysis:

  • Use immunohistochemistry to map At3g05165 localization in different tissues

  • Determine if protein redistribution occurs during water stress

  • Identify cell types that upregulate the transporter during drought

• Protein Interaction Networks:

  • Immunoprecipitate At3g05165 during normal and drought conditions

  • Identify differential interaction partners using mass spectrometry

  • Construct stress-responsive protein networks

• Translational Research:

  • Test antibody cross-reactivity with crop homologs

  • Use successful antibodies to screen germplasm collections for optimal expression patterns

  • Correlate protein abundance with drought tolerance traits

These approaches have yielded insights into how sugar transport and allocation contribute to drought resilience in model plants, with potential applications in crop improvement .

What methods can be used to quantitatively assess At3g05165 protein dynamics during developmental transitions?

To quantitatively track At3g05165 protein changes during plant development:

Quantitative Assessment Techniques:

• Multiplexed Western Blotting:

  • Use dual-color detection systems with At3g05165 antibody and control protein antibody

  • Normalize signal to loading controls like actin or GAPDH

  • Apply digital image analysis for precise quantification

• Quantitative ELISA:

  • Develop standard curves using recombinant At3g05165 protein

  • Process developmental samples in parallel

  • Calculate absolute protein concentrations

• Mass Spectrometry-Based Quantification:

  • Implement Selected Reaction Monitoring (SRM) or Parallel Reaction Monitoring (PRM)

  • Use stable isotope-labeled peptides as internal standards

  • Target unique peptides identified by the antibody's epitope mapping

• High-Content Imaging:

  • Perform immunostaining with At3g05165 antibody across developmental stages

  • Apply automated image analysis for quantitative assessment

  • Generate heat maps of expression intensity in different tissues and cell types

These methods have successfully tracked sugar transporter dynamics during seed development, germination, and flowering transitions in Arabidopsis, revealing stage-specific regulation patterns .

How do results from At3g05165 antibody studies compare with transcriptomic data for the same gene?

Understanding the relationship between transcript and protein levels is crucial:

Comparative Analysis Framework:

Integration Strategy:

• Perform time-course experiments with parallel sampling for RNA and protein extraction

• Compare transcript kinetics (via qRT-PCR) with protein accumulation (via quantitative Western blotting)

• Calculate correlation coefficients between mRNA and protein levels

• Identify time lags between transcriptional induction and protein accumulation

• Investigate discrepancies through analysis of protein stability, translational efficiency, or post-translational regulation

Research has shown that At3g05165 transcript and protein levels are not always perfectly correlated, particularly during stress responses, indicating significant post-transcriptional regulation that can only be detected through protein-level studies with specific antibodies .

What are emerging applications of At3g05165 antibodies in understanding plant membrane trafficking?

At3g05165 antibodies are becoming valuable tools for studying membrane trafficking pathways:

Emerging Applications:

• Super-Resolution Microscopy:

  • Use At3g05165 antibodies combined with photoactivatable fluorophores

  • Track single-molecule dynamics in living cells

  • Achieve nanometer-scale resolution of transporter clustering and movement

• Proximity Labeling:

  • Combine At3g05165 antibodies with enzymatic tags (BioID, APEX)

  • Identify proteins in close proximity within the membrane environment

  • Map the spatiotemporal dynamics of transporter-associated protein complexes

• Correlative Light and Electron Microscopy:

  • Detect At3g05165 by immunofluorescence, then prepare the same sample for electron microscopy

  • Precisely localize transporters in the context of subcellular ultrastructure

  • Resolve membrane microdomains housing sugar transporters

• Optogenetic Control:

  • Use antibodies to validate the location and function of optogenetically-controlled At3g05165 variants

  • Study real-time transporter dynamics in response to environmental stimuli

These advanced techniques are revealing unprecedented details about how sugar transporters like At3g05165 are trafficked, regulated, and organized within plant cell membranes .

How might At3g05165 antibodies contribute to synthetic biology approaches in plants?

At3g05165 antibodies can play significant roles in plant synthetic biology:

Applications in Synthetic Biology:

• Protein Scaffold Validation:

  • Use antibodies to confirm correct assembly of synthetic protein complexes incorporating At3g05165

  • Verify proper membrane integration of engineered transporters

  • Quantify expression levels of synthetic constructs

• Biosensor Development:

  • Create split-antibody complementation systems for monitoring protein-protein interactions

  • Develop FRET-based biosensors using At3g05165 antibody fragments

  • Design antibody-based reporters for sugar transport activity

• Metabolic Engineering Verification:

  • Confirm overexpression of At3g05165 in engineered high-yield crops

  • Track protein localization in cells modified for enhanced sugar transport

  • Assess stability of modified transporters under field conditions

• Protein Evolution Studies:

  • Use antibodies to screen libraries of mutagenized At3g05165 variants

  • Identify mutations affecting stability, trafficking, or function

  • Apply directed evolution approaches to develop transporters with novel properties

These antibody-dependent approaches are accelerating the development of plants with improved sugar allocation, stress resistance, and yield potential through precise modification of sugar transport systems .