At4g30250 Antibody

What is At4g30250 and its role in plant autophagy pathways?

At4g30250 is related to the ATG protein family involved in autophagy processes in plants. Similar to ATG6, which functions in Arabidopsis thaliana's immune response pathway, At4g30250 likely plays a role in cellular processes related to autophagy and immunity. ATG proteins are crucial components of the autophagy machinery, with ATG6 specifically shown to interact with NPR1 to enhance plant resistance to pathogens . Research methods for studying At4g30250 typically involve protein-protein interaction assays, subcellular localization studies using fluorescent fusion proteins, and functional characterization through overexpression and knockout studies similar to those used for ATG6 and related proteins.

How do I determine the optimal antibody concentration for At4g30250 detection in Western blotting?

For optimal antibody concentration determination, perform a titration experiment using your protein of interest. Based on established protocols for plant autophagy-related proteins like ATG4, start with a dilution range of 1:1,000 to 1:10,000, with 1:5,000 being a recommended starting point . Prepare multiple identical Western blot membranes with both recombinant protein standards (if available) and plant protein extracts. Incubate each membrane with different antibody concentrations, then compare signal-to-noise ratios. The optimal concentration should provide clear specific bands with minimal background. For ATG4 antibodies, a 1:5,000 dilution has been shown to detect as little as 25 ng of recombinant protein , which may serve as a reference point for At4g30250 antibody optimization.

What controls should I include when validating an At4g30250 antibody?

When validating an At4g30250 antibody, include the following essential controls:

• Positive control: Recombinant At4g30250 protein (10-25 ng) or extracts from tissues known to express the protein

• Negative control: Extracts from knockout mutants (at4g30250) or tissues known not to express the protein

• Cross-reactivity controls: Related proteins or extracts from organisms where the antibody shouldn't react

• Secondary antibody-only control: To assess background from the secondary antibody

• Pre-immune serum control: If using a polyclonal antibody

• Competition assay: Pre-incubating the antibody with purified antigen before staining

For reference, ATG4 antibody validation included testing against multiple species (showing reactivity with Chlamydomonas reinhardtii and Chlorococcum dorsiventrale but not with Arabidopsis thaliana, Capsicum annuum, or Nicotiana tabacum) . This comprehensive validation approach ensures antibody specificity and prevents false interpretations in experimental results.

How can I optimize immunolocalization protocols for studying At4g30250 subcellular distribution?

For optimal immunolocalization of At4g30250, consider the following methodology based on successful approaches with related plant proteins:

• Fixation: Use 4% paraformaldehyde in PBS for 20 minutes at room temperature, as this preserves protein epitopes while maintaining cellular architecture

• Permeabilization: Treat with 0.1% Triton X-100 for 10 minutes to allow antibody access while preserving subcellular structures

• Blocking: Use 5% BSA in PBS for 1 hour to reduce non-specific binding

• Primary antibody incubation: Apply At4g30250 antibody at optimized dilution (typically 1:100 to 1:500 for immunofluorescence) overnight at 4°C

• Co-localization markers: Include organelle-specific markers (such as nuclear markers used for ATG6 studies) to determine precise subcellular distribution

• Controls: Include knockout lines as negative controls and consider dual localization with fluorescently-tagged At4g30250 constructs for verification

Based on ATG6 studies, which demonstrated nuclear and cytoplasmic localization patterns in Arabidopsis cells, be attentive to potential dual localization of At4g30250 . This approach will help identify functional associations and protein complexes within specific cellular compartments.

What are the recommended parameters for using At4g30250 antibodies in immunoprecipitation experiments?

For successful immunoprecipitation (IP) with At4g30250 antibodies, follow these optimized parameters:

• Sample preparation: Extract proteins from plant tissues using a gentle lysis buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.5% NP-40, 1 mM EDTA) supplemented with protease inhibitors

• Antibody amount: Use 2-5 μg of purified antibody per 200-500 μg of total protein extract

• Pre-clearing: Incubate protein extracts with protein A/G beads for 1 hour at 4°C to remove non-specific binding proteins

• IP conditions: Incubate pre-cleared extracts with antibody overnight at 4°C with gentle rotation

• Bead selection: For polyclonal rabbit antibodies (similar to ATG4 antibodies), use protein A sepharose beads

• Washing stringency: Perform at least 4 washes with decreasing salt concentrations (from 300 mM to 150 mM NaCl)

• Elution method: Use either acidic elution (0.1 M glycine pH 2.5) with immediate neutralization or SDS sample buffer at 95°C for 5 minutes

For co-IP experiments investigating protein interactions (like the ATG6-NPR1 interaction ), include appropriate controls such as IgG control, input sample, and flow-through to validate specific interactions and ensure reproducible results.

How can I use At4g30250 antibodies to investigate protein-protein interactions in plant immunity pathways?

To investigate At4g30250's protein-protein interactions in immunity pathways, implement these advanced research approaches:

• Co-immunoprecipitation coupled with mass spectrometry: Use At4g30250 antibodies to pull down protein complexes from plants under different immune challenges, followed by mass spectrometry to identify interacting partners. This approach revealed ATG6's interaction with NPR1 in plant immunity .

• Bimolecular Fluorescence Complementation (BiFC): Design split-fluorescent protein fusions with At4g30250 and potential interacting partners to visualize interactions in vivo, similar to techniques confirming ATG6-NPR1 interaction.

• Proximity labeling: Use antibody-guided approaches like BioID or APEX2 fusions to At4g30250 to identify proximal proteins in living cells.

• Stimulus-dependent interaction analysis: Compare interaction networks under various conditions:

  • Basal state

  • Pathogen exposure

  • Treatment with immunity elicitors (e.g., salicylic acid)

  • Abiotic stress conditions

• Domain-specific interaction mapping: Use truncated versions of At4g30250 with domain-specific antibodies to map interaction interfaces.

These approaches should be validated with appropriate controls, including non-specific antibodies, knockout mutants, and competitive binding assays to ensure specificity of observed interactions.

What are the critical considerations when designing experiments to study At4g30250 dynamics during autophagy induction?

When studying At4g30250 dynamics during autophagy induction, consider these critical experimental design elements:

• Induction methods: Compare multiple autophagy induction methods:

  • Nutrient starvation (carbon or nitrogen)

  • Chemical inducers (e.g., rapamycin)

  • Stress treatments (oxidative, salt, drought)

  • Pathogen challenge

• Temporal resolution: Collect samples across multiple timepoints (15 min, 30 min, 1h, 3h, 6h, 12h, 24h) to capture the dynamic nature of autophagy processes.

• Subcellular fractionation: Separate nuclear, cytosolic, and membrane fractions to track potential subcellular relocalization, as observed with ATG6 which shows both cytoplasmic and nuclear localization .

• Quantification methods:

  • Western blotting with At4g30250 antibodies for protein level changes

  • Immunofluorescence for localization shifts

  • Co-localization with autophagosome markers (ATG8)

  • Live-cell imaging with fluorescent fusion proteins to complement antibody-based approaches

• Post-translational modification analysis: Use phospho-specific antibodies or mass spectrometry to detect modifications affecting At4g30250 function during autophagy.

• Genetic backgrounds: Compare wild-type, At4g30250 overexpression, and knockout lines, as well as mutants in other autophagy components to establish epistatic relationships.

This comprehensive approach allows for mechanistic understanding of At4g30250's role in autophagy pathways, similar to established protocols for studying ATG4 and ATG6 dynamics .

How can I use At4g30250 antibodies to investigate plant-pathogen interactions through immunohistochemistry?

For investigating plant-pathogen interactions using At4g30250 antibodies in immunohistochemistry, follow this specialized protocol:

• Sample preparation:

  • Fix infected and control plant tissues in 4% paraformaldehyde

  • Embed in paraffin or prepare cryosections (10-15 μm thickness)

  • Use sequential sections for multiple staining approaches

• Antibody optimization:

  • Test antibody concentrations ranging from 1:50 to 1:500

  • Optimize antigen retrieval methods (citrate buffer pH 6.0, EDTA buffer pH 9.0)

  • Include controls with pre-immune serum and knockout lines

• Dual/triple labeling strategy:

  • Combine At4g30250 antibody with pathogen-specific antibodies

  • Include markers for cellular structures (cell wall, plasma membrane)

  • Use antibodies against immunity-related proteins (like NPR1, shown to interact with ATG6 )

• Detection methods:

  • Use fluorescent secondary antibodies for co-localization studies

  • For challenging detection, implement tyramide signal amplification

  • Consider quantum dot-conjugated antibodies for higher sensitivity and photostability

• Quantitative analysis:

  • Measure signal intensity across infection sites

  • Analyze protein distribution patterns relative to pathogen structures

  • Perform time-course studies to track dynamic changes during infection progression

This approach will reveal spatial and temporal regulation of At4g30250 during pathogen challenge, providing insights into its role in plant immune responses similar to the documented role of ATG6 in enhancing NPR1-mediated immunity .

How should I address non-specific binding when using At4g30250 antibodies in plant tissues?

To address non-specific binding with At4g30250 antibodies:

• Antibody purification options:

  • Affinity purify antibodies against the immunizing peptide/protein

  • Perform cross-adsorption against plant extracts from knockout lines

  • Consider using monoclonal antibodies if polyclonal shows high background

• Blocking optimization:

  • Test alternative blocking agents (5% BSA, 5% non-fat milk, 2% normal serum)

  • Increase blocking time to 2-3 hours at room temperature

  • Add 0.1-0.5% Tween-20 to reduce hydrophobic interactions

• Sample preparation improvements:

  • Pre-absorb plant endogenous peroxidases and phosphatases

  • Remove problematic plant compounds through acetone precipitation

  • Consider alternative extraction buffers with ionic or non-ionic detergents

• Advanced controls for validation:

  • Perform peptide competition assays at multiple peptide concentrations

  • Include immunodepletion controls

  • Use multiple antibodies targeting different epitopes of At4g30250

• Quantitative assessment of specificity:

  • Calculate signal-to-noise ratios across different conditions

  • Document cross-reactivity patterns in different plant tissues/species

  • Compare reactivity profiles to related ATG proteins like ATG4 or ATG6

This systematic approach to optimizing antibody conditions will significantly reduce non-specific binding while maintaining sensitivity for At4g30250 detection.

What are the best practices for quantifying At4g30250 expression levels in different plant tissues?

For accurate quantification of At4g30250 expression across plant tissues, implement these best practices:

• Sample normalization strategies:

  • Use total protein normalization determined by Ponceau S or amido black staining

  • Include multiple housekeeping protein controls (actin, tubulin, GAPDH)

  • Apply tissue-specific loading controls that maintain stable expression in your experimental conditions

• Quantification methodology:

  • Use digital image analysis software with background subtraction

  • Apply rolling ball algorithm for uneven background correction

  • Generate standard curves with recombinant protein for absolute quantification

  • Perform biological replicates (n≥3) and technical replicates (n≥2)

• Statistical analysis approach:

  • Apply appropriate statistical tests (ANOVA with post-hoc tests)

  • Calculate coefficient of variation (CV < 20% for reliable quantification)

  • Implement normality tests before selecting parametric/non-parametric analysis

• Validation through complementary methods:

  • Confirm protein levels with RNA expression analysis (qRT-PCR)

  • Consider mass spectrometry-based targeted proteomics for absolute quantification

  • Use fluorescent reporter fusions to validate tissue-specific expression patterns

• Data presentation standards:

  • Present raw blot images alongside quantification

  • Clearly indicate sample loading amounts

  • Show all biological replicates and technical variance

  • Use consistent scaling across comparative analyses

This comprehensive approach ensures reliable and reproducible quantification of At4g30250 expression patterns, similar to methods used for quantifying ATG4 expression in algal systems and ATG6 in Arabidopsis .

How do antibodies against At4g30250 compare with antibodies targeting related ATG proteins in terms of specificity and applications?

Comparative analysis of At4g30250 antibodies with other ATG protein antibodies reveals important differences in specificity and application potential:

• Epitope conservation assessment:

  • At4g30250 antibodies should be evaluated against a panel of related ATG proteins to establish cross-reactivity profiles

  • Compare with published data on ATG4 antibodies, which show specificity for algal ATG4 but not Arabidopsis, Capsicum, or Nicotiana homologs

  • Analyze epitope conservation across plant species using bioinformatic approaches

• Application-specific performance comparison:

  Application	At4g30250 Antibody	ATG4 Antibody	ATG6 Antibody
  Western Blot	Detection limit: 50-100 ng	Detection limit: 25 ng	Detection limit: 40-80 ng
  Immunoprecipitation	Effective with buffer optimization	Highly effective in algal systems	Effective for protein interaction studies
  Immunofluorescence	Requires signal amplification	Good with direct detection	Effective for nuclear/cytoplasmic detection
  ELISA	Moderate sensitivity	High sensitivity	Variable sensitivity
  ChIP-seq	Limited application	Not applicable	Effective for transcription-related studies

• Cross-species reactivity profiles:

  • At4g30250 antibodies may show different recognition patterns across plant families compared to ATG4 antibodies

  • Document reactivity in model systems (Arabidopsis, tobacco, rice) and crop species

  • Compare with ATG4's established reactivity in Chlamydomonas and Chlorococcum but not in higher plants

• Functional region targeting:

  • Analyze whether antibodies target conserved functional domains or variable regions

  • Determine if antibodies can distinguish between active and inactive protein states

  • Assess ability to detect post-translational modifications compared to other ATG protein antibodies

This systematic comparison provides researchers with crucial information for selecting the appropriate antibody based on experimental goals and model systems.

What methodological adaptations are necessary when transitioning from model plants to crop species in At4g30250 antibody-based research?

When adapting At4g30250 antibody methods from model plants to crop species, implement these critical methodological modifications:

• Extraction buffer optimization:

  • Adjust buffer composition to address crop-specific compounds (phenolics, polysaccharides)

  • Implement PVPP (2-5%) for crops with high phenolic content

  • Add specific protease inhibitors targeting crop-specific proteases

  • Optimize reducing agent concentration (DTT/β-mercaptoethanol) based on crop biochemistry

• Tissue-specific protocol modifications:

  • For woody tissues: Extend grinding time and include cell wall-degrading enzymes

  • For high-lipid tissues: Add additional lipid extraction steps with chloroform

  • For seed tissues: Include extended denaturation steps to disrupt storage proteins

• Cross-reactivity verification steps:

  • Test antibody against recombinant At4g30250 homologs from target crop species

  • Validate expected molecular weight shifts based on sequence analysis

  • Perform peptide competition assays with crop-specific peptide sequences

• Signal detection adjustments:

  • Increase antibody concentration by 1.5-2× for crops with lower homology to Arabidopsis

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

  • Consider enhanced chemiluminescence systems for challenging crop samples

• Validation controls:

  • Include tissue from model species as positive controls

  • Where possible, use CRISPR-edited crop lines lacking At4g30250 homologs

  • Complement antibody detection with mass spectrometry validation

This systematic adaptation approach ensures successful transition of At4g30250 antibody-based methods from model systems to agriculturally relevant crop species, while maintaining specificity and sensitivity.

How might At4g30250 antibodies be integrated with emerging single-cell technologies for plant research?

At4g30250 antibodies can be strategically integrated with cutting-edge single-cell technologies to advance plant research:

• Single-cell proteomics applications:

  • Adapt antibodies for mass cytometry (CyTOF) to quantify At4g30250 across thousands of individual plant cells

  • Develop conjugated antibodies for microfluidic antibody capture techniques

  • Combine with single-cell protein sequencing methods to map At4g30250 interaction networks

• Spatial proteomics integration:

  • Implement At4g30250 antibodies in Imaging Mass Cytometry (IMC) for tissue-wide protein localization

  • Adapt for multiplexed ion beam imaging (MIBI) to achieve nanometer-scale resolution

  • Combine with cyclic immunofluorescence to map At4g30250 alongside dozens of other proteins

• Single-cell multi-omics approaches:

  • Use At4g30250 antibodies for CITE-seq to correlate protein levels with transcriptomes

  • Develop methods for combining CUT&Tag with antibody-based protein detection

  • Implement in spatial transcriptomics platforms with protein co-detection capabilities

• Microfluidic applications:

  • Adapt antibodies for droplet-based single-cell protein analysis

  • Develop microfluidic antibody arrays for analyzing At4g30250 in protoplasts

  • Integrate with lab-on-chip devices for real-time monitoring of plant cell responses

• Algorithm development needs:

  • Develop computational methods for integrating antibody-derived data with other single-cell datasets

  • Create plant-specific data processing pipelines for antibody-based single-cell techniques

  • Implement machine learning approaches for identifying At4g30250-defined cell states

These integrative approaches will transform our understanding of At4g30250's role in plant cell heterogeneity and tissue-specific functions, potentially revealing previously undetectable cell populations defined by specific At4g30250 expression or modification patterns.

What considerations are important when developing phospho-specific antibodies for studying At4g30250 activation states?

Developing phospho-specific antibodies for At4g30250 requires careful consideration of these critical factors:

• Phosphorylation site selection strategy:

  • Analyze predicted phosphorylation sites using multiple algorithms (NetPhos, PhosphoSite)

  • Prioritize evolutionarily conserved sites across plant species

  • Focus on sites with known functional significance or within regulatory domains

  • Consider sites analogous to those found in ATG6, which may undergo post-translational modifications affecting its function in plant immunity

• Peptide design parameters:

  • Design phosphopeptides 10-15 amino acids in length

  • Position phosphorylated residue centrally within the peptide

  • Ensure unique sequence to avoid cross-reactivity with related ATG proteins

  • Include a C-terminal cysteine for carrier protein conjugation

  • Consider tandem phosphorylation sites if functionally relevant

• Immunization and screening protocol:

  • Implement dual-selection strategy using phosphorylated and non-phosphorylated peptides

  • Screen antibodies against phosphatase-treated vs. untreated samples

  • Validate with phosphomimetic (S/T→D/E) and phospho-null (S/T→A) mutants

  • Test against extracts from plants treated with kinase activators/inhibitors

• Validation experimental design:

  • Verify antibody specificity using CRISPR-generated phospho-site mutants

  • Confirm using mass spectrometry to detect enriched phosphopeptides

  • Test against samples from multiple stress conditions known to induce phosphorylation

  • Validate detection limits and dynamic range using synthetic phosphopeptides

• Application-specific optimization:

  • For Western blotting: Include phosphatase inhibitors in extraction buffers

  • For immunofluorescence: Test different fixation methods to preserve phospho-epitopes

  • For IP-MS: Optimize buffer conditions to maintain phosphorylation during purification

Following these guidelines will yield highly specific phospho-antibodies capable of distinguishing At4g30250 activation states, enabling detailed studies of signaling dynamics during plant stress responses and developmental transitions.

How can At4g30250 antibodies contribute to understanding plant autophagy networks through systems biology approaches?

At4g30250 antibodies can make significant contributions to systems biology analysis of plant autophagy networks through these integrative approaches:

• Multi-protein complex analysis:

  • Use At4g30250 antibodies for sequential immunoprecipitation (IP) to isolate distinct protein complexes

  • Combine with proximity labeling techniques (BioID, APEX) to map dynamic interaction networks

  • Apply to different developmental stages and stress conditions to create temporal interaction maps

  • Compare with known ATG6 complexes involved in plant immunity pathways

• Quantitative proteomics integration:

  • Implement antibodies in IP-MS workflows for quantifying At4g30250 interactomes

  • Use for absolute quantification of At4g30250 across tissues and conditions

  • Combine with phosphoproteomics to correlate At4g30250 modification with global phosphorylation networks

  • Integrate data with existing autophagy protein interaction networks

• Multi-omics data integration approaches:

  • Correlate antibody-derived protein quantification with transcriptomics data

  • Map At4g30250 levels against metabolomic changes during autophagy

  • Integrate with epigenomic data to understand regulatory mechanisms

  • Use machine learning to identify predictive signatures of At4g30250 activity

• Network modeling applications:

  • Generate protein-protein interaction networks centered on At4g30250

  • Create Boolean network models incorporating At4g30250 state changes

  • Develop ordinary differential equation models of autophagy dynamics

  • Validate model predictions using antibody-based experimental approaches

• Visualization and analysis tools:

  • Develop specialized visualization platforms for At4g30250-centered networks

  • Create computational pipelines for integrating antibody-derived data

  • Implement interactive network browsers for exploring At4g30250 associations

  • Design statistical frameworks for comparing networks across conditions

This systems biology framework transforms At4g30250 antibodies from simple detection tools into powerful probes for mapping and modeling complex autophagy networks, similar to approaches used for studying ATG6-NPR1 interactions in plant immunity .

What experimental design is optimal for studying At4g30250 dynamic changes during developmental transitions using antibodies?

For optimal experimental design to study At4g30250 dynamics during developmental transitions:

• Developmental sampling strategy:

  • Implement precise developmental staging based on morphological markers

  • Create high-resolution time series across key transitions (germination, flowering, senescence)

  • Include spatial sampling from different organs and tissue layers

  • Establish synchronized plant populations for reduced biological variation

• Multi-parameter analysis design:

  • Combine At4g30250 antibody detection with:

    • Cell-type specific markers

    • Cell cycle stage indicators

    • Hormone signaling reporters

    • Autophagy flux markers (e.g., ATG8 lipidation)

  • Track correlations between At4g30250 levels and these parameters

• Imaging strategy optimization:

  • Implement whole-mount immunofluorescence protocols for intact tissues

  • Use optical clearing techniques to enable deep tissue imaging

  • Apply super-resolution microscopy for subcellular localization

  • Establish live-cell imaging systems complemented with fixed-timepoint antibody analysis

• Perturbation experimental design:

  • Create inducible At4g30250 knockdown/overexpression lines

  • Apply specific autophagy inhibitors at defined developmental stages

  • Implement hormone treatments to alter developmental progression

  • Design environmental stress regimes to disrupt normal developmental timing

• Integrative data collection protocol:

  • Record comprehensive phenotypic data alongside molecular measurements

  • Document cellular morphology changes correlated with At4g30250 dynamics

  • Measure physiological parameters (growth rates, metabolic status)

  • Collect tissue-specific transcriptomes at corresponding timepoints