At1g77650 Antibody

What is At1g77650 (ATG6) and why are antibodies against it valuable in plant research?

At1g77650 encodes ATG6, a critical protein in Arabidopsis thaliana that plays dual roles in autophagy and plant immunity. Recent studies have revealed that ATG6 directly interacts with NPR1 (NON-EXPRESSOR OF PATHOGENESIS-RELATED GENES 1), a key immune regulator, significantly enhancing Arabidopsis resistance to pathogens like Pseudomonas syringae pv. tomato DC3000/avrRps4 . ATG6 overexpression increases nuclear accumulation of NPR1 and promotes the formation of SA-induced NPR1 condensates (SINCs) . Antibodies against ATG6 are invaluable tools for studying these protein-protein interactions, subcellular localization patterns, and expression levels during immune responses. They enable researchers to track ATG6's involvement in both autophagy and defense signaling pathways through various immunological techniques.

What techniques can be used to validate At1g77650 antibody specificity?

Validating antibody specificity is crucial for reliable experimental results. For At1g77650 antibodies, a comprehensive validation workflow should include:

• Genetic knockout controls: Testing the antibody in ATG6 knockout lines versus wild-type plants. This genetic approach has proven superior to orthogonal approaches for immunofluorescence applications, with studies showing 80% confirmation rate for antibodies validated through genetic strategies versus only 38% for orthogonal methods .

• Western blot analysis: Confirming single-band detection at the expected molecular weight (~68 kDa for ATG6) in wild-type samples and absence in knockout lines. Studies show that well-performing antibodies should specifically immunodetect their target protein without non-specific bands .

• Immunoprecipitation efficiency: Verifying that the antibody can successfully enrich its target from plant extracts, as approximately 37% of antibodies without manufacturer recommendations for IP may still effectively enrich their target antigens .

• Mass spectrometry confirmation: Analyzing immunoprecipitated proteins to confirm ATG6 enrichment and identify potential cross-reactive proteins.

• Immunofluorescence patterns: Comparing localization patterns with published ATG6-fluorescent protein fusion studies, noting that only 39% of commercially recommended antibodies succeed in immunofluorescence applications .

All validation data should be documented systematically to ensure reproducibility.

What are the recommended applications for At1g77650 antibodies?

At1g77650 (ATG6) antibodies can be utilized in multiple experimental applications:

Importantly, antibodies should be tested in all intended applications regardless of manufacturer recommendations, as studies have shown that companies sometimes under-recommend successful products, with 37% of antibodies without IP recommendations still successfully enriching their target antigens .

How should At1g77650 antibodies be stored and handled?

Proper storage and handling of At1g77650 antibodies is critical for maintaining their specificity and sensitivity:

• Storage temperature: Store antibody aliquots at -20°C for long-term storage and at 4°C for short-term use (1-2 weeks).

• Aliquoting: Divide stock solutions into single-use aliquots to avoid repeated freeze-thaw cycles, which can lead to degradation and aggregation. Each freeze-thaw cycle can potentially affect protein recognition.

• Buffer considerations: For diluted working solutions, use PBS or TBS with 0.05% sodium azide as a preservative. For applications studying ATG6-NPR1 interactions, avoid reducing agents that might disrupt protein structure .

• Concentration monitoring: Track antibody concentration and activity over time. A gradual increase in required antibody concentration may indicate declining activity.

• Contamination prevention: Use sterile technique when handling antibodies to prevent microbial growth.

• Documentation: Keep detailed records of storage conditions, thawing dates, and application-specific performance over time.

Properly stored and handled antibodies will provide more consistent results across experiments, particularly important when studying dynamic processes like ATG6's role in plant immunity.

How can At1g77650 antibodies be used to study its interaction with NPR1?

At1g77650 (ATG6) antibodies are powerful tools for investigating ATG6-NPR1 interactions that enhance plant immunity. Research has demonstrated that ATG6 directly interacts with NPR1, facilitating its nuclear accumulation and activating defense responses . Several methodological approaches using antibodies can elucidate this interaction:

• Co-immunoprecipitation (Co-IP): ATG6 antibodies can pull down ATG6-NPR1 complexes, confirming their physical interaction in vivo. Studies have shown ATG6 interacts with NPR1 in the nucleus, as demonstrated through fluorescence microscopy .

• Proximity ligation assay (PLA): This technique can visualize ATG6-NPR1 interactions in situ, providing spatial information about where these interactions occur within plant cells.

• Chromatin immunoprecipitation (ChIP): Using ATG6 antibodies for ChIP can help determine whether ATG6 associates with NPR1 at specific promoter regions of defense genes like PR1 and PR5, whose expression is significantly enhanced when ATG6 and NPR1 work together .

• Immunofluorescence microscopy: Dual immunolabeling with ATG6 and NPR1 antibodies can track their co-localization during pathogen challenge, particularly the enhanced nuclear accumulation of NPR1 observed in ATG6-overexpressing plants .

• Western blot analysis: Sequential immunoblotting can monitor how ATG6 affects NPR1 protein levels and stability, as ATG6 has been shown to increase NPR1 protein levels and promote SINC formation .

When designing these experiments, it's essential to include appropriate controls and standardize conditions to account for SA-induced changes in both proteins' behaviors.

What epitope considerations are important when selecting At1g77650 antibodies?

Selecting antibodies with appropriate epitope recognition is crucial for studying At1g77650 (ATG6) function:

• Functional domain awareness: ATG6 contains several functional domains involved in protein-protein interactions. Antibodies targeting regions required for NPR1 interaction may interfere with this binding in vitro. Research has shown that ATG6 directly interacts with NPR1, so epitope selection should consider preserving these interaction sites .

• Post-translational modifications (PTMs): Choose antibodies whose epitopes avoid known or potential phosphorylation, ubiquitination, or other modification sites that may be regulatory during immune responses.

• Conservation analysis: For cross-species studies, select antibodies targeting evolutionarily conserved epitopes. This is particularly important when comparing ATG6 functions across different plant species.

• Structural accessibility: Consider whether the epitope is accessible in the native protein conformation. Surface-exposed epitopes generally yield better results in immunoprecipitation and immunofluorescence applications.

• Paralog specificity: Ensure the antibody specifically recognizes ATG6 and not related proteins in the ATG family to avoid cross-reactivity.

For optimal results in studying ATG6-NPR1 interactions, polyclonal antibodies raised against multiple epitopes may provide advantages for capturing different conformational states that occur during immune activation.

How do different fixation methods affect At1g77650 antibody performance in immunofluorescence?

Fixation methods significantly impact At1g77650 (ATG6) antibody performance in immunofluorescence studies:

• Paraformaldehyde fixation: Standard 4% PFA preserves protein localization but may mask epitopes. For ATG6, which localizes to both cytoplasm and nucleus during immune responses, this fixation maintains cellular architecture while potentially limiting antibody accessibility.

• Methanol fixation: Provides better epitope exposure but disrupts membrane structures. This may be problematic when studying ATG6's role in autophagosome formation but beneficial for visualizing nuclear localization during NPR1 interaction .

• Glutaraldehyde-based fixation: Offers stronger protein cross-linking but significantly increases autofluorescence, which can mask the true signal in plant tissues.

• Combined fixation approaches: Sequential paraformaldehyde/methanol fixation can balance structural preservation with epitope accessibility.

Research shows that only 39% of antibodies recommended for immunofluorescence perform successfully in standardized validation protocols , highlighting the critical importance of fixation optimization. When studying ATG6-NPR1 interactions, different fixation methods should be systematically compared, as these proteins relocalize between cytoplasm and nucleus during immune responses. Proper fixation is especially important for preserving SA-induced NPR1 condensates (SINCs), which ATG6 has been shown to stabilize .

What are the challenges in detecting native levels of At1g77650 protein?

Detecting native levels of At1g77650 (ATG6) protein presents several challenges:

• Low endogenous expression: ATG6 often exists at low basal levels, increasing primarily during immune responses or autophagy induction. This requires highly sensitive detection methods and careful signal amplification.

• Protein turnover dynamics: ATG6 may undergo dynamic regulation during defense responses, with rapid changes in protein stability that complicate consistent detection.

• Subcellular redistribution: ATG6 redistributes between cytoplasmic and nuclear compartments during immune activation , necessitating careful fractionation protocols for subcellular localization studies.

• Cross-reactivity concerns: Even validated antibodies may exhibit some cross-reactivity with related ATG family proteins, requiring stringent controls.

• Post-translational modifications: Various stress conditions might trigger modifications that alter antibody recognition. Recent findings show ATG6 interacts with NPR1 to increase its stability and nuclear accumulation , suggesting potential regulatory modifications.

• Sample preparation variability: Plant tissue disruption efficiency varies between samples, affecting protein extraction yield.

To overcome these challenges, researchers should:

• Use genetic knockout controls for specificity verification

• Employ signal enhancement methods like TSA (tyramide signal amplification)

• Carefully optimize extraction buffers to preserve protein integrity

• Consider mass spectrometry-based validation for confirmation

• Compare multiple antibodies when possible, as studies show significant variability in antibody performance

How can At1g77650 antibodies be utilized to investigate ATG6's dual role in autophagy and plant immunity?

At1g77650 (ATG6) antibodies can be strategically deployed to investigate the protein's dual functionality:

• Differential complex analysis: Immunoprecipitation with ATG6 antibodies followed by mass spectrometry can identify distinct protein complexes associated with either autophagy (e.g., ATG8) or immunity (e.g., NPR1). Recent research has demonstrated ATG6's direct interaction with NPR1, enhancing plant immunity against pathogens like Pst DC3000/avrRps4 .

• Temporal dynamics monitoring: Western blotting with ATG6 antibodies during pathogen infection time courses can track protein level changes and post-translational modifications. ATG6 overexpression significantly increases NPR1 protein levels and nuclear accumulation , suggesting regulation at the protein stability level.

• Pathway-specific localization: Immunofluorescence under conditions that specifically induce either autophagy or immune responses can map ATG6 redistribution patterns. During immune responses, ATG6 facilitates NPR1 nuclear accumulation .

• Chromatin association studies: ChIP with ATG6 antibodies can determine whether ATG6 associates with defense gene promoters alongside NPR1. Studies show ATG6 promotes expression of NPR1 downstream target genes like PR1 and PR5 .

• Developmental context analysis: Immunohistochemistry across different plant tissues and developmental stages can reveal tissue-specific functions of ATG6.

• Stress-specific modifications: Immunoprecipitation under different stress conditions (nutrient limitation versus pathogen exposure) followed by post-translational modification analysis can identify condition-specific regulatory mechanisms.

This multi-faceted approach can clarify how one protein orchestrates both cellular recycling through autophagy and defense signaling through NPR1 interaction.

What controls should be included when using At1g77650 antibodies?

Robust controls are essential for experiments using At1g77650 (ATG6) antibodies:

• Genetic controls:

  • ATG6 knockout/knockdown lines as negative controls

  • ATG6 overexpression lines as positive controls

  • Wild-type plants as reference standards

  • Studies show genetic validation approaches provide more reliable antibody characterization than orthogonal approaches

• Technical controls:

  • Primary antibody omission to assess secondary antibody non-specific binding

  • Isotype controls to evaluate non-specific binding

  • Pre-immune serum controls for polyclonal antibodies

  • Pre-absorption with immunizing peptide to confirm epitope specificity

• Biological condition controls:

  • SA treatment samples for studying ATG6-NPR1 interactions, as SA promotes NPR1 nuclear translocation

  • Pathogen-challenged samples (e.g., Pst DC3000/avrRps4) to induce ATG6-NPR1 immune response

  • Autophagy induction conditions (e.g., nutrient starvation) to assess ATG6's autophagy function

• Cross-validation controls:

  • Multiple antibodies targeting different epitopes of ATG6

  • Correlation with ATG6-fluorescent protein fusion localization patterns

  • Comparison with mRNA expression data

• Application-specific controls:

  • For co-IP: IgG control immunoprecipitations

  • For ChIP: Input samples and IgG controls

  • For immunofluorescence: Autofluorescence controls

Implementing these controls helps distinguish true signals from artifacts, particularly important given that studies show 31% of published work using antibodies for Western blot employed products that failed independent validation .

How can At1g77650 antibodies be used in co-immunoprecipitation to identify interaction partners?

At1g77650 (ATG6) antibodies can be effectively used in co-immunoprecipitation (co-IP) to discover and validate protein interactions:

• Optimized extraction conditions:

  • Use gentle, non-denaturing buffers (typically HEPES or Tris-based, pH 7.4-7.6)

  • Include protease inhibitors to prevent degradation

  • Consider detergent selection carefully (0.5-1% NP-40 or Triton X-100 typically maintains interactions)

  • For studying ATG6-NPR1 interactions, include 1mM EDTA and 1mM DTT to maintain protein stability

• Antibody immobilization strategies:

  • Direct coupling to protein A/G beads

  • Biotinylated antibodies with streptavidin support

  • Pre-formed antibody-bead complexes to minimize background

• Reciprocal confirmation approach:

  • Primary co-IP: ATG6 antibody to pull down complexes, probe for partners like NPR1

  • Reciprocal co-IP: NPR1 antibody to pull down complexes, probe for ATG6

  • This bidirectional validation strengthens interaction claims, as demonstrated in research showing ATG6-NPR1 interaction

• Elution techniques:

  • Gentle elution with excess immunizing peptide

  • Standard SDS-based elution for stronger interactions

  • Native elution using low pH for maintaining complex integrity

• Interaction verification methods:

  • Western blot analysis of co-IP samples (most common)

  • Mass spectrometry for unbiased partner identification

  • Functional validation of identified interactions through genetic studies

Research has shown that ATG6 directly interacts with NPR1, and this interaction increases Arabidopsis resistance to pathogens . When planning co-IP experiments, consider that only about 37% of antibodies without manufacturer recommendations for IP successfully enrich their target proteins , highlighting the importance of validation.

What are the optimal conditions for western blotting with At1g77650 antibodies?

Optimal western blotting conditions for At1g77650 (ATG6) antibodies require careful protocol optimization:

• Sample preparation:

  • Extract proteins using buffer containing 50mM Tris-HCl (pH 7.5), 150mM NaCl, 1% Triton X-100, 0.1% SDS, and protease inhibitors

  • For nuclear protein extraction, use specialized nuclear extraction buffers to properly assess ATG6 nuclear accumulation during immune responses

  • Fresh sample preparation yields better results than frozen samples

• Gel electrophoresis parameters:

  • 8-10% polyacrylamide gels provide optimal resolution for ATG6 (~68 kDa)

  • Load 20-50 μg total protein per lane

  • Include positive controls (ATG6 overexpression) and negative controls (atg6 mutant)

• Transfer conditions:

  • Semi-dry transfer: 15V for 30-45 minutes

  • Wet transfer: 100V for 1 hour at 4°C

  • PVDF membranes typically yield better results than nitrocellulose for ATG6 detection

• Blocking parameters:

  • 5% non-fat dry milk in TBST (most common)

  • For phospho-specific detection, 5% BSA in TBST

  • Block for 1 hour at room temperature or overnight at 4°C

• Antibody incubation:

  • Primary antibody dilution: 1:1000 to 1:5000 (requires optimization)

  • Incubation: Overnight at 4°C with gentle agitation

  • Secondary antibody: 1:5000 to 1:10000, 1 hour at room temperature

• Detection system:

  • Enhanced chemiluminescence (ECL) for standard detection

  • Fluorescent secondary antibodies for quantitative analysis

  • Exposure times typically 30 seconds to 5 minutes

For studies of ATG6-NPR1 interactions, consider dual probing to simultaneously detect both proteins or sequential probing with stripping between detections. Research has shown that ATG6 increases NPR1 protein levels under both normal and SA treatment conditions , making quantitative western blotting particularly valuable.

How can At1g77650 antibodies be used for chromatin immunoprecipitation studies?

Chromatin immunoprecipitation (ChIP) with At1g77650 (ATG6) antibodies requires specific considerations:

• Chromatin preparation:

  • Crosslink plant tissue with 1% formaldehyde for 10-15 minutes under vacuum

  • Quench with 0.125M glycine for 5 minutes

  • Use sonication to generate 200-500 bp DNA fragments

  • Verify fragmentation efficiency by agarose gel electrophoresis

• Immunoprecipitation optimization:

  • Pre-clear chromatin with protein A/G beads to reduce background

  • Use 2-5 μg of ATG6 antibody per immunoprecipitation

  • Include IgG control and input samples (10% of starting material)

  • Incubate overnight at 4°C with gentle rotation

• Washing and elution protocols:

  • Use increasingly stringent wash buffers to reduce non-specific binding

  • Elute protein-DNA complexes with elution buffer containing 1% SDS and 0.1M NaHCO₃

  • Reverse crosslinks by incubation at 65°C for 4-6 hours

  • Treat with proteinase K and RNase A before DNA purification

• Target gene selection:

  • Focus on NPR1-regulated defense genes like PR1 and PR5, as ATG6 has been shown to promote their expression

  • Include ICS1 gene regions, as ATG6 overexpression increases ICS1 expression

  • Test multiple primer pairs spanning promoter and coding regions

• Analysis methods:

  • qPCR for targeted analysis of specific loci

  • ChIP-seq for genome-wide binding profile

  • Compare binding patterns under normal conditions versus pathogen challenge or SA treatment

While ATG6 has not been traditionally considered a DNA-binding protein, recent research showing its interaction with NPR1 and its impact on defense gene expression suggests potential chromatin association, either directly or as part of regulatory complexes that include NPR1.