At1g64760 Antibody

What is At1g64760 and why is it important in plant research?

At1g64760 encodes the ATG6 protein in Arabidopsis thaliana, which plays critical roles in plant immunity and autophagy pathways. Recent research indicates that ATG6 directly interacts with NPR1 (Nonexpresser of Pathogenesis-Related genes 1), a key immune regulator, significantly enhancing plant resistance to pathogens such as Pseudomonas syringae . The antibody against this protein is essential for studying plant immune responses, autophagy mechanisms, and stress responses in Arabidopsis.

How should At1g64760 (ATG6) antibody be validated before experimental use?

Validation of At1g64760 antibody should follow the "five pillars" of antibody characterization:

• Genetic strategies: Use ATG6 knockout/knockdown lines as negative controls

• Orthogonal strategies: Compare antibody-dependent results with antibody-independent methods

• Multiple independent antibody strategies: Use different antibodies targeting distinct epitopes of ATG6

• Recombinant strategies: Test with overexpressed ATG6 protein systems

• Immunocapture MS strategies: Verify captured proteins using mass spectrometry

At minimum, researchers should document that the antibody: (i) binds specifically to the ATG6 protein; (ii) maintains specificity in complex protein mixtures; (iii) shows minimal cross-reactivity with other proteins; and (iv) performs consistently under the specific experimental conditions being used .

What are the recommended applications for At1g64760 antibody in plant research?

At1g64760 (ATG6) antibody is suitable for multiple applications including:

• Western blotting to detect ATG6 protein levels

• Immunoprecipitation to study ATG6 interactions (particularly with NPR1)

• Immunofluorescence to visualize subcellular localization

• Chromatin immunoprecipitation if studying DNA-binding complexes involving ATG6

• Co-immunoprecipitation to verify protein-protein interactions

The antibody is particularly valuable for studying ATG6's role in promoting nuclear accumulation of NPR1 and formation of SA-induced NPR1 condensates (SINCs) .

How should researchers evaluate At1g64760 antibody specificity?

Researchers should employ multiple complementary approaches to verify antibody specificity:

It's critical to document all validation steps in publications to enhance reproducibility .

What analytical methods are recommended for characterizing ATG6-NPR1 interactions?

To properly characterize ATG6-NPR1 interactions, researchers should employ:

• Co-immunoprecipitation using At1g64760 antibody to pull down protein complexes

• Reciprocal co-IP with NPR1 antibody

• Proximity ligation assays to visualize interactions in situ

• Bimolecular fluorescence complementation for in vivo interaction confirmation

• Nuclear/cytoplasmic fractionation followed by immunoblotting to quantify NPR1 nuclear accumulation

These methods can verify that "ATG6 directly interacts with NPR1" and "ATG6 overexpression significantly increased nuclear accumulation of NPR1" .

How should researchers design experiments to study ATG6's effect on NPR1 nuclear localization?

When studying ATG6's impact on NPR1 nuclear localization, implement this experimental framework:

• Generate and validate transgenic lines:

  • ATG6 overexpression lines

  • atg6 knockout/knockdown lines

  • NPR1-reporter fusions (e.g., NPR1-GFP)

• Design appropriate treatments:

  • Mock treatment

  • Pathogen challenge (e.g., Pst DC3000/avrRps4)

  • Salicylic acid treatment at various concentrations

• Cellular fractionation protocol:

  • Separate nuclear and cytoplasmic fractions

  • Quantify NPR1 in each fraction via western blotting

  • Use appropriate loading controls (nuclear and cytoplasmic markers)

• Microscopy validation:

  • Perform confocal microscopy with NPR1-fluorescent protein fusions

  • Quantify nuclear/cytoplasmic fluorescence ratios

This approach provides multiple lines of evidence for ATG6's role in NPR1 nuclear accumulation.

What controls are essential when using At1g64760 antibody in immunoprecipitation experiments?

Essential controls for At1g64760 antibody immunoprecipitation include:

• Input sample: To verify protein presence before IP

• Negative controls:

  • IgG control: Non-specific antibody of same isotype

  • Knockout/knockdown tissue: Material lacking the target protein

  • Pre-immune serum: When using polyclonal antibodies

• Blocking peptide control: Pre-incubation with the immunizing peptide

• Reciprocal IP: Pull-down with antibody against interacting partner (e.g., NPR1)

• Denaturing controls: Testing interactions under denaturing conditions to distinguish direct vs. indirect interactions

These controls help distinguish specific signals from background and verify true protein-protein interactions.

How can researchers employ At1g64760 antibody to investigate ATG6's role in SA-induced NPR1 condensate formation?

To investigate ATG6's role in SA-induced NPR1 condensate (SINC) formation:

• Prepare experimental systems:

  • Wild-type, ATG6-overexpression, and atg6 mutant plants

  • Treatment conditions: control vs. SA treatment at defined concentrations

• Immunofluorescence approach:

  • Fix tissue samples with appropriate fixatives (e.g., 4% PFA)

  • Permeabilize tissues to allow antibody access

  • Co-stain with At1g64760 (ATG6) and NPR1 antibodies

  • Use confocal microscopy to visualize co-localization in nuclear condensates

• Biochemical approach:

  • Perform nuclear isolation with varying salt concentrations

  • Analyze condensate properties through separation techniques

  • Immunoblot fractions to identify ATG6 and NPR1 co-segregation

• Live-cell imaging:

  • Generate fluorescent protein fusions for both proteins

  • Monitor condensate dynamics in real-time after SA treatment

These approaches can verify that "ATG6 can stabilize NPR1 and promote the formation of SINCs (SA-induced NPR1 condensates)-like condensates" .

What advanced techniques can be used to track the dynamics of ATG6-NPR1 interactions during pathogen response?

Advanced techniques for tracking ATG6-NPR1 interaction dynamics include:

• Single-molecule tracking:

  • Tag ATG6 and NPR1 with photoconvertible fluorophores

  • Monitor real-time movement and interaction in living cells

• FRET/FLIM analysis:

  • Generate appropriate donor-acceptor fluorophore pairs

  • Measure energy transfer efficiency as indicator of molecular proximity

  • Quantify interaction changes over time post-pathogen exposure

• Optogenetic approaches:

  • Create light-inducible ATG6 variants

  • Trigger ATG6 activation at precise timepoints

  • Monitor subsequent NPR1 relocalization

• Super-resolution microscopy:

  • Use STED, PALM, or STORM techniques

  • Resolve subcellular structures below diffraction limit

  • Precisely map interaction domains within nuclear compartments

These techniques provide temporal and spatial resolution beyond conventional methods, revealing dynamic aspects of ATG6-NPR1 interactions during immune responses.

How should researchers address inconsistent results when using At1g64760 antibody across different experimental conditions?

When facing inconsistent results with At1g64760 antibody:

• Systematically assess variables:

  • Antibody batch variation: Test multiple lots

  • Sample preparation: Standardize extraction buffers, protease inhibitors

  • Experimental conditions: Temperature, incubation times, buffer composition

  • Tissue specificity: Different plant tissues may require optimized protocols

• Validate antibody performance:

  • Re-test antibody specificity under your specific conditions

  • Consider epitope masking effects in different sample preparations

  • Evaluate potential post-translational modifications affecting epitope recognition

• Establish positive controls:

  • Include samples with known high ATG6 expression

  • Consider using recombinant ATG6 protein as standard

• Document all protocol variations:

  • Maintain detailed records of conditions that yield consistent results

  • Report relevant methodological details in publications

Addressing variability through systematic troubleshooting enhances experimental reproducibility.

What statistical approaches are recommended for analyzing ATG6-NPR1 co-localization data?

For rigorous analysis of ATG6-NPR1 co-localization data:

• Quantitative co-localization metrics:

  • Pearson's correlation coefficient: Measures linear correlation between fluorescence intensities

  • Manders' overlap coefficient: Determines proportion of pixels with both signals

  • Object-based approaches: Count discrete structures showing both proteins

• Appropriate statistical testing:

  • Compare coefficients across experimental conditions using ANOVA or appropriate non-parametric alternatives

  • Perform multiple comparison corrections (e.g., Bonferroni, Tukey HSD)

  • Report effect sizes alongside p-values

• Sample size considerations:

  • Analyze multiple cells across independent biological replicates

  • Power analysis to determine minimum required measurements

  • Report sample sizes transparently

• Control analyses:

  • Test for random co-localization by image randomization

  • Account for chromatic aberrations using technical controls

  • Include non-interacting protein pairs as negative controls

This statistical rigor prevents overinterpretation of coincidental co-localization and provides confidence in genuine biological interactions.

How might new antibody technologies enhance studies of At1g64760 (ATG6) function in plant immunity?

Emerging antibody technologies that could advance ATG6 research include:

• Engineered "sweeping antibodies" with pH-dependent binding:

  • Antibodies engineered with pH-dependent antigen binding similar to receptor-ligand interactions

  • Enhanced FcRn binding at neutral pH for improved plasma/cellular clearance

  • Potential to achieve 50-1000 fold greater antigen reduction compared to conventional antibodies

• Single-domain antibodies (nanobodies):

  • Smaller size allows access to previously inaccessible cellular compartments

  • Can be expressed intracellularly as "intrabodies" to track ATG6 in living cells

  • Potential for higher specificity to particular ATG6 conformational states

• Proximity-labeling antibody conjugates:

  • ATG6 antibodies conjugated to enzymes like BioID or APEX2

  • Enables identification of transient interaction partners in native contexts

  • Could reveal previously unknown components of ATG6-NPR1 complexes

These technologies could reveal new insights into the temporal and spatial dynamics of ATG6's role in plant immunity.