At5g17750 Antibody

What is the function of ATG5 (At5g17750) in plant autophagy mechanisms?

ATG5 (Autophagy-related protein 5) plays a crucial role in plant autophagy pathways. The protein forms a conjugate with ATG12 that is essential for plant nutrient recycling processes. In Arabidopsis thaliana, ATG5 is involved in two primary functions: complete proteolysis of chloroplast stroma proteins in senescent leaves and the degradation of damaged peroxisomes . This conjugation is a critical step in autophagosome formation, where the ATG5-ATG12 conjugate forms a cup-shaped isolation membrane that detaches from the membrane immediately before or after autophagosome formation is completed . Unlike in mammalian systems where ATG5 is also implicated in apoptotic processes, plant ATG5 appears primarily dedicated to autophagic functions related to nutrient recycling and organelle quality control.

How should researchers select appropriate antibodies for plant ATG5 detection?

When selecting antibodies for plant ATG5 detection, researchers should consider:

• Specificity validation: Confirm that the antibody specifically recognizes plant ATG5 and not just recombinant proteins. Some antibodies may recognize recombinant ATG5 but require additional validation for endogenous protein detection .

• Conjugate detection capability: Determine whether the antibody can detect both free ATG5 (~32 kDa) and the ATG5-ATG12 complex (~55 kDa), which may be important depending on your research questions .

• Cross-reactivity profile: Verify whether the antibody shows cross-reactivity with ATG proteins from other species if conducting comparative studies. Some antibodies are raised against specific species (e.g., Arabidopsis thaliana) and may not recognize homologs in other plant species .

• Application compatibility: Ensure the antibody is validated for your specific application (Western blot, immunoprecipitation, etc.). For example, the antibody described in the search results is recommended at 1:1000 dilution for Western blot applications .

What are the optimal storage conditions for maintaining ATG5 antibody efficacy?

To maintain antibody efficacy for plant ATG5 research:

• Storage temperature: Store lyophilized/reconstituted antibodies at -20°C to maintain stability .

• Aliquoting strategy: Once reconstituted, make small aliquots to avoid repeated freeze-thaw cycles that can degrade antibody performance .

• Reconstitution protocol: For lyophilized antibodies, reconstitute with the recommended volume of sterile water (e.g., 50 μl for some preparations) .

• Sample preparation: Prior to opening stored antibodies, briefly spin the tubes to collect all material that might adhere to the cap or sides of the tube .

• Preservative options: Some antibodies can be provided with preservatives like ProClin upon request, which may extend shelf-life while maintaining performance characteristics .

How can researchers validate ATG5 antibody specificity in plant systems?

Validating antibody specificity in plant systems requires a multi-step approach:

• Recombinant protein controls: Test antibody against purified recombinant ATG5 protein to establish baseline recognition .

• Knockout validation: Compare antibody reactivity between wild-type plants and atg5 knockout mutants. The antibody signal should be absent in the knockout line.

• Cross-reactivity assessment: Systematically test against related ATG proteins (e.g., ATG7) to confirm absence of cross-reactivity .

• Size verification: Confirm that the detected protein bands match the expected molecular weights: ~32 kDa for free ATG5 and ~55 kDa for the ATG5-ATG12 complex .

• Epitope mapping: For detailed characterization, determine which specific region of the protein is recognized by the antibody. For example, some antibodies are raised against specific amino acid sequences (like 28-275 aa in human ATG5) .

What methodological approaches are recommended for isolating ATG5-interacting proteins in plant systems?

For isolating ATG5-interacting proteins in plant systems:

• Co-immunoprecipitation optimization:

  • Use mild lysis buffers (e.g., 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 1% NP-40) supplemented with protease inhibitors

  • Pre-clear lysates with protein A/G beads before immunoprecipitation

  • Incubate plant lysates with anti-ATG5 antibody overnight at 4°C

  • Capture complexes using protein A/G magnetic beads

  • Perform stringent washing steps (4-5 times) while maintaining sufficient buffer volumes

• Cross-linking approaches:

  • Utilize in vivo cross-linking with 1-2% formaldehyde to stabilize transient interactions

  • For increased specificity, use cleavable cross-linkers that allow reversal during analysis

• Proximity labeling techniques:

  • Express ATG5 fused to BioID or TurboID in Arabidopsis

  • Apply biotin for defined time periods to label proximal proteins

  • Isolate biotinylated proteins using streptavidin beads

  • Identify interacting partners via mass spectrometry

• Validation strategies:

  • Confirm interactions using reverse co-immunoprecipitation

  • Verify physiological relevance through mutant studies and localization analysis

How do isolation and analysis methods differ between detecting free ATG5 versus ATG5-ATG12 conjugates?

The detection and analysis of free ATG5 versus ATG5-ATG12 conjugates requires different methodological considerations:

When analyzing the conjugate formation:

• Use non-reducing conditions to preserve the conjugate when appropriate

• Include both positive controls (nitrogen starvation to induce autophagy) and negative controls (atg7 mutants that cannot form the conjugate)

• Consider using antibodies that specifically recognize the junction region between ATG5 and ATG12 for conjugate-specific detection

What are the emerging computational approaches for analyzing antibody-antigen interactions in plant autophagy research?

Recent advances in computational modeling have improved analysis of antibody-antigen interactions:

• Systems serology approaches:

  • Employ experimental techniques to dissect antibodies' features and functions

  • Apply computational methods to analyze datasets and understand interconnected relationships between profiled antibodies and immune system response

  • Simplify complex molecular interactions to identify patterns in antibody effectiveness

• Pattern recognition algorithms:

  • Identify signature patterns in antibody binding and neutralization profiles

  • Create visual representations showing correlations between antibody structures and target antigens

  • Use color-depth coding to represent strength of correlations in multidimensional analyses

• Epitope mapping prediction:

  • Utilize machine learning algorithms to predict epitope regions on plant ATG proteins

  • Compare predicted epitopes with experimentally verified binding sites

  • Generate heat maps showing probability of epitope regions across the ATG5 protein sequence

• Structural modeling integration:

  • Combine antibody binding data with protein structural models of plant ATG5

  • Simulate conformational changes during ATG5-ATG12 conjugation

  • Predict accessibility of epitopes during different stages of autophagosome formation

How can researchers troubleshoot non-specific binding issues in ATG5 immunodetection experiments?

When encountering non-specific binding in ATG5 immunodetection:

• Blocking optimization:

  • Test different blocking agents (5% non-fat milk, 3-5% BSA, commercial blocking buffers)

  • Extend blocking time to 2-3 hours at room temperature or overnight at 4°C

  • Add 0.1-0.3% Tween-20 to blocking and antibody incubation buffers

• Antibody dilution titration:

  • Perform systematic dilution series (1:500 to 1:5000) to determine optimal concentration

  • For plant ATG5 detection, starting with 1:1000 dilution is recommended based on validated protocols

  • Consider overnight incubation at 4°C instead of shorter room temperature incubation

• Sample preparation refinement:

  • Improve protein extraction methods specific to plant tissues

  • Include additional centrifugation steps to remove debris

  • Add protease inhibitors immediately during extraction

  • Consider using specialized plant protein extraction buffers containing PVP to remove phenolic compounds

• Validation controls:

  • Include atg5 knockout plants as negative controls

  • Use recombinant ATG5 protein as positive control

  • Perform peptide competition assays to confirm specificity

• Cross-adsorption technique:

  • Pre-incubate antibody with plant extract from atg5 mutants

  • Remove antibodies binding to non-specific targets

  • Use the pre-adsorbed antibody solution for detection

What experimental design factors should be considered when studying ATG5 in stress response pathways?

When designing experiments to study ATG5 in plant stress responses:

• Stress induction protocols:

  • Nitrogen starvation: Transfer plants to nitrogen-free medium for 2-7 days

  • Carbon starvation: Maintain plants in darkness for 2-5 days

  • Oxidative stress: Apply H₂O₂ (1-10 mM) or methyl viologen (1-50 μM)

  • Salt stress: Apply NaCl (100-200 mM) treatments

• Tissue sampling timeline:

  • Collect samples at multiple time points (0, 6, 12, 24, 48, 72 hours)

  • Include recovery phase samples after stress removal

  • Consider diurnal variations in autophagy rates

• Appropriate controls:

  • Include atg5, atg7, and atg12 mutant lines

  • Use both constitutive autophagy markers and stress-responsive controls

  • Maintain identical growth conditions between experimental and control plants

• Multi-assay approach:

  • Combine Western blot analysis of ATG5-ATG12 conjugate levels

  • Visualize autophagosomes using fluorescent marker lines (GFP-ATG8)

  • Quantify autophagic flux using inhibitors like concanamycin A

  • Assess physiological parameters (chlorophyll content, ion leakage, ROS levels)

How can researchers optimize protein extraction methods specifically for ATG5 detection in different plant tissues?

Optimizing protein extraction for ATG5 detection across plant tissues:

• Tissue-specific extraction buffers:

• Critical extraction parameters:

  • Maintain cold chain throughout extraction (4°C)

  • Use protease inhibitor cocktail with broad spectrum coverage

  • Include phosphatase inhibitors when studying regulated autophagy

  • Perform extraction in sufficient buffer volume (5-10 ml per gram tissue)

• Tissue disruption methods:

  • For leaves: Mortar and pestle grinding in liquid nitrogen

  • For roots: Bead beating with 1.0-2.0 mm ceramic beads

  • For recalcitrant tissues: Cryogenic grinding with dry ice

• Post-extraction processing:

  • Centrifuge at ≥20,000 × g for 15 minutes at 4°C

  • Filter supernatant through 0.45 μm filter for highly fibrous tissues

  • Consider additional TCA/acetone precipitation for samples with interfering compounds

What methodological approaches can resolve contradictory results between ATG5 protein levels and autophagic activity?

When faced with contradictory results between ATG5 protein levels and observed autophagy:

• Multi-marker validation approach:

  • Analyze multiple autophagy markers (ATG8-PE, NBR1 degradation)

  • Compare results from different methodologies (Western blot, microscopy, proteomics)

  • Assess autophagic flux rather than single time-point measurements

• Regulatory mechanism investigation:

  • Examine post-translational modifications of ATG5

  • Analyze subcellular localization of ATG5 using fractionation or microscopy

  • Investigate potential compensatory pathways in atg5 mutant backgrounds

• Experimental condition standardization:

  • Precisely control plant age, growth conditions, and stress application

  • Harvest tissues at consistent times to account for diurnal variation

  • Use internal standards for protein quantification

• Quantitative analysis framework:

  • Apply densitometry to quantify Western blot band intensities

  • Calculate the ratio of ATG5-ATG12 conjugate to free ATG5

  • Perform statistical analysis across biological replicates (n≥3)

• Causality assessment:

  • Conduct time-course experiments to establish temporal relationships

  • Use inducible expression systems to manipulate ATG5 levels

  • Apply pharmacological inhibitors of specific autophagy steps

How are new antibody engineering approaches improving specificity in plant autophagy research?

Recent antibody engineering approaches have enhanced specificity in plant research:

• Single-chain variable fragment (scFv) technology:

  • Development of plant-specific scFvs that maintain specificity while providing better tissue penetration

  • Expression of intrabodies that can track ATG5 localization in living plant cells

  • Creation of bispecific antibodies that simultaneously detect ATG5 and its interaction partners

• Phage display selection:

  • Isolation of highly specific plant ATG5 binders through iterative selection processes

  • Enrichment for antibodies that distinguish between free ATG5 and the ATG5-ATG12 conjugate

  • Selection under varying pH and salt conditions to ensure stability in plant extraction buffers

• Computational antibody design:

  • In silico prediction of optimal binding regions specific to plant ATG5

  • Molecular dynamics simulations to optimize antibody-antigen interactions

  • Structure-guided affinity maturation to improve binding properties

• Site-specific conjugation methods:

  • Development of site-specifically labeled antibodies for quantitative imaging

  • Creation of antibody-fluorophore conjugates optimized for plant cell imaging

  • Production of antibody-enzyme fusions for proximity-based detection systems

What are the latest developments in using recombinant ATG5 for antibody characterization and screening?

Recent developments in recombinant ATG5 production for antibody work include:

• Expression system optimization:

  • Plant-based expression systems for proper folding and post-translational modifications

  • Cell-free protein synthesis for rapid production of ATG5 variants

  • Bacterial expression with solubility tags (MBP, SUMO) to improve yield

• Structural variants production:

  • Generation of defined ATG5 fragments for epitope mapping

  • Production of pre-formed ATG5-ATG12 conjugates as reference standards

  • Creation of ATG5 mutants lacking specific functional domains

• Assay development approaches:

  • Surface plasmon resonance (SPR) with immobilized recombinant ATG5

  • Bio-layer interferometry for real-time antibody binding kinetics

  • Multiplex bead-based assays for high-throughput antibody screening

• Validation strategies:

  • Competitive binding assays using recombinant ATG5 versus native plant extracts

  • Pull-down experiments with tagged recombinant ATG5 as positive controls

  • Cross-validation between different recombinant forms (prokaryotic vs. eukaryotic expression)

How can researchers integrate ATG5 antibody-based detection with other autophagy monitoring techniques?

Integration of antibody-based detection with complementary techniques:

• Combined fluorescence approaches:

  • Correlative light and electron microscopy (CLEM) with immunogold labeling of ATG5

  • Co-localization studies using fluorescently-tagged ATG8 and antibody-detected ATG5

  • Super-resolution microscopy to resolve autophagosome formation sites

• Multi-omics integration:

  • Correlation of ATG5 protein levels with transcriptomics data

  • Integration of ATG5 interactome data with global proteomics

  • Metabolomic profiling to link autophagy activity with cellular metabolic state

• Flux measurement combination:

  • Tandem mRFP-GFP-ATG8 reporter systems complemented with ATG5 quantification

  • Autophagy substrate degradation rates correlated with ATG5-ATG12 conjugate levels

  • Use of lysosomal inhibitors to assess flux while monitoring ATG5 dynamics

• Temporal analysis framework:

  • Time-lapse imaging with immunofluorescence at defined intervals

  • Pulse-chase experiments combined with ATG5 quantification

  • Inducible systems to trigger autophagy while monitoring ATG5 recruitment kinetics