At1g71250 Antibody

What is AT1G71250 and why is it significant in plant research?

AT1G71250 is a gene located on chromosome 1 of Arabidopsis thaliana (thale cress) that encodes a GDSL-like Lipase/Acylhydrolase superfamily protein . This protein belongs to a diverse family of enzymes that hydrolyze ester bonds and often play roles in plant development, stress responses, and pathogen defense. The protein has alternative identifiers including F3I17.10 and F3I17_10 . Research significance stems from its potential involvement in plant immunity pathways, possibly similar to mechanisms observed with other Arabidopsis proteins like ATG6 and NPR1, which have been shown to interact and enhance plant resistance to pathogens .

What approaches are most effective for generating AT1G71250-specific antibodies?

Generating specific antibodies against AT1G71250 requires careful antigen design. The most effective approach utilizes recombinant protein expression systems with the full-length AT1G71250 protein or specific domains using available cDNA ORF clones . For optimal epitope selection, researchers should analyze the protein sequence to identify unique regions that distinguish it from other GDSL-like family members. Immunization protocols should include multiple boosts to ensure high-titer antibody production. Post-production validation must include Western blot analysis using both wildtype Arabidopsis tissue and at1g71250 knockout lines to confirm specificity.

How does AT1G71250 antibody validation differ from other plant protein antibodies?

AT1G71250 antibody validation presents unique challenges compared to antibodies against more abundant plant proteins. A comprehensive validation approach should include:

What are the optimal protein extraction methods for immunodetection of AT1G71250?

The GDSL-like lipase encoded by AT1G71250 presents specific extraction challenges due to potential membrane association and enzymatic activity. The optimal extraction protocol includes:

• Harvest fresh Arabidopsis tissue (100-200 mg) and flash-freeze in liquid nitrogen

• Grind tissue to a fine powder while maintaining freezing temperatures

• Add extraction buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 10% glycerol, 1% Triton X-100, 1 mM EDTA, 5 mM DTT, 1× protease inhibitor cocktail)

• Include specific lipase inhibitors (PMSF at 1 mM) to prevent self-degradation

• Homogenize and incubate with gentle rotation at 4°C for 30 minutes

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

• Collect supernatant and quantify protein concentration
  This approach has shown reliable performance for downstream antibody-based applications and helps maintain protein integrity during extraction.

How can immunoprecipitation with AT1G71250 antibodies be optimized to study protein interactions?

Immunoprecipitation (IP) optimization for AT1G71250 should follow methods similar to those used to demonstrate protein interactions in Arabidopsis immunity research . The recommended protocol includes:

• Use a gentle lysis buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.5% NP-40, 1 mM EDTA, protease inhibitors) to preserve protein-protein interactions

• Pre-clear lysate with Protein A/G beads for 1 hour at 4°C to reduce non-specific binding

• Optimize antibody concentration (typically 2-5 μg per 1 mg total protein) and incubation time (overnight at 4°C)

• Include appropriate controls: IgG control, input sample, and when possible, tissue from at1g71250 mutants

• For elution, use either gentle conditions (low pH glycine buffer) for downstream functional assays or denaturing conditions (SDS buffer) for maximum recovery

• Verify interactions through both Western blot and mass spectrometry analysis
  This methodology has successfully identified protein-protein interactions in similar plant immunity studies and can be adapted specifically for AT1G71250 research .

What approaches are most effective for quantifying AT1G71250 protein levels in different physiological conditions?

Accurate quantification of AT1G71250 protein across different conditions requires careful methodological considerations:

• Western blot quantification:

  • Use digital image capture systems rather than film for linear dynamic range

  • Apply densitometry analysis with appropriate software (ImageJ, Image Lab)

  • Include standard curves with known amounts of recombinant AT1G71250

  • Run at least three biological replicates per condition

• Normalization strategies:

  • Primary: housekeeping proteins (actin, tubulin)

  • Secondary: total protein staining (Ponceau S, SYPRO Ruby)

  • Analyze ratios of AT1G71250 to reference protein or total protein

• Advanced quantification using targeted mass spectrometry:

  • Develop Selected Reaction Monitoring (SRM) or Parallel Reaction Monitoring (PRM) assays

  • Use isotope-labeled peptide standards for absolute quantification

  • Select AT1G71250-specific peptides that ionize well and are unique to the protein
    This multi-method approach provides robust quantification across different experimental conditions and treatments.

How can AT1G71250 antibodies be used to investigate subcellular localization changes during stress responses?

Understanding AT1G71250 protein dynamics during stress responses requires sophisticated localization studies. The recommended approach includes:

• Subcellular fractionation:

  • Separate nuclei, cytosol, membrane, and organelle fractions

  • Confirm fraction purity with compartment-specific markers

  • Analyze AT1G71250 distribution across fractions before and after stress treatment

  • Compare with known stress-responsive proteins like NPR1, which shows stress-induced nuclear accumulation

• Immunofluorescence microscopy:

  • Fix tissues at multiple timepoints following stress application

  • Use optimized antigen retrieval methods to maximize signal

  • Co-stain with organelle markers and DNA stains

  • Perform Z-stack imaging for 3D distribution analysis

• Quantification methods:

  • Measure nuclear/cytoplasmic signal ratios

  • Track formation of protein condensates or bodies similar to SA-induced NPR1 condensates

  • Apply appropriate statistical analysis to demonstrate significance of relocalization
    This approach can reveal whether AT1G71250 undergoes dynamic relocalization during stress responses, similar to the increased nuclear accumulation of NPR1 observed during immune responses .

What mass spectrometry approaches are recommended for characterizing post-translational modifications of AT1G71250?

Comprehensive characterization of AT1G71250 post-translational modifications (PTMs) requires sophisticated mass spectrometry approaches similar to those used for antibody characterization :

How can AT1G71250 antibodies be integrated with CRISPR-Cas9 gene editing to study protein function?

Combining antibody-based approaches with CRISPR-Cas9 gene editing creates powerful research opportunities:

• Generating and validating epitope-tagged AT1G71250 variants:

  • Design CRISPR constructs to introduce small epitope tags (FLAG, HA, V5) at the AT1G71250 genomic locus

  • Create point mutations in functional domains while preserving epitope recognition

  • Validate tagged variants with both epitope antibodies and AT1G71250-specific antibodies

• Functional domain analysis:

  • Create precise deletions or mutations in predicted functional domains

  • Use AT1G71250 antibodies to assess protein expression and stability

  • Compare wildtype and mutant protein interactions and localization

• Validation workflow:

  • Confirm genomic edits by sequencing

  • Verify protein expression by Western blot

  • Assess functional consequences through established assays
    This integrated approach allows precise correlation between genetic modifications and protein-level consequences, enabling structure-function studies of the AT1G71250 protein.

How should researchers resolve contradictory results when using different AT1G71250 antibodies?

Contradictory results with different AT1G71250 antibodies require systematic investigation:

• Epitope mapping analysis:

  • Determine which regions of AT1G71250 are recognized by each antibody

  • Assess potential epitope masking by protein interactions or modifications

  • Test antibodies against recombinant fragments to confirm binding sites

• Methodological investigations:

  • Compare extraction methods and their effects on protein conformation

  • Evaluate fixation protocols for immunohistochemistry applications

  • Test different blocking agents to reduce non-specific binding

• Systematic validation approach:

  • Create a validation matrix testing all antibodies against the same positive/negative controls

  • Document exact protocols used with each antibody

  • Consider independent methods (MS, fluorescent tagging) to resolve contradictions

• Biological explanations:

  • Investigate potential protein isoforms or truncations

  • Consider developmental or stress-induced modifications

  • Evaluate tissue-specific expression patterns
    This systematic approach helps distinguish between technical artifacts and genuine biological phenomena when interpreting contradictory antibody results.

What are common causes of false negatives when detecting AT1G71250, and how can these be addressed?

False negatives in AT1G71250 detection often stem from several methodological challenges:

How can researchers distinguish between specific and non-specific signals when using AT1G71250 antibodies?

Distinguishing specific from non-specific signals requires rigorous controls and validation:

• Essential controls:

  • Genetic controls: at1g71250 knockout/knockdown lines should show no or reduced signal

  • Absorption controls: pre-incubation with purified antigen should eliminate specific signal

  • Secondary antibody-only controls: identify background from secondary antibody

• Signal validation methods:

  • Compare signals across multiple antibodies targeting different epitopes

  • Verify expected molecular weight on Western blots

  • Confirm expected subcellular localization pattern

  • Validate with orthogonal methods (MS identification, fluorescent protein tagging)

• Quantitative assessment:

  • Calculate signal-to-noise ratios across different tissues and conditions

  • Document antibody dilution optimization experiments

  • Perform titration experiments to determine optimal antibody concentrations
    These validation approaches are essential for ensuring that observed signals genuinely represent AT1G71250 protein rather than experimental artifacts.

How might AT1G71250 function relate to established plant immunity pathways involving ATG6 and NPR1?

While direct evidence linking AT1G71250 to ATG6-NPR1 immunity pathways is limited, its GDSL-like lipase activity suggests potential roles:

• Hypothesized functional connections:

  • Membrane remodeling during immune responses, potentially affecting signaling platforms

  • Production of lipid-derived defense signals during pathogen challenge

  • Possible involvement in SA-dependent pathways, similar to NPR1-dependent responses

• Methodological approaches to investigate connections:

  • Co-immunoprecipitation with AT1G71250 antibodies followed by Western blot for ATG6 or NPR1

  • Genetic interaction studies crossing at1g71250 mutants with immunity pathway mutants

  • Comparative protein expression analysis under conditions that induce NPR1 accumulation

  • Assessment of PR gene expression (PR1, PR5) in at1g71250 mutants compared to controls

• Protein localization studies:

  • Determine if AT1G71250 forms condensates similar to SA-induced NPR1 condensates

  • Assess whether AT1G71250 localization changes during immune responses

  • Investigate if ATG6 overexpression affects AT1G71250 protein levels or localization
    These approaches could reveal whether AT1G71250 functions within established immunity pathways or represents a distinct mechanism in plant defense.

What experimental approaches can determine if AT1G71250 undergoes dynamic changes during pathogen infection similar to NPR1?

Investigating AT1G71250 dynamics during pathogen infection requires temporal and spatial analysis:

• Time-course protein analysis:

  • Collect samples at multiple timepoints (0, 3, 6, 12, 24, 48 hours) after pathogen inoculation

  • Perform Western blot quantification of total AT1G71250 protein levels

  • Compare dynamics with known immunity markers like NPR1, which shows increased accumulation during immune responses

• Subcellular distribution analysis:

  • Perform cell fractionation to isolate nuclear, cytoplasmic, and membrane fractions

  • Quantify AT1G71250 distribution across fractions before and after infection

  • Assess whether AT1G71250 forms condensates similar to NPR1's SINCs-like structures

• Protein stability assessment:

  • Conduct cycloheximide chase assays to measure protein half-life

  • Compare stability in wildtype vs. immunity pathway mutants

  • Determine if stability changes during immune responses

• Correlation with defense responses:

  • Monitor AT1G71250 levels in relation to SA accumulation and PR gene expression

  • Assess whether pathogen-induced cell death differs between wildtype and at1g71250 mutants
    These approaches would determine whether AT1G71250 undergoes dynamic changes similar to the increased protein levels and nuclear accumulation observed for NPR1 during immune responses .

How can mass spectrometry be optimized to detect AT1G71250 interactions with immunity-related proteins?

Detecting AT1G71250 protein interactions through mass spectrometry requires specialized approaches:

• Sample preparation optimization:

  • Use crosslinking agents (DSP, formaldehyde) to stabilize transient interactions

  • Employ gentle extraction conditions to preserve protein complexes

  • Perform immunoprecipitation with AT1G71250 antibodies under native conditions

  • Include appropriate controls (IgG pulldown, knockout tissue IP)

• MS acquisition strategies:

  • Apply data-independent acquisition (DIA) for comprehensive detection

  • Use high-resolution MS for accurate identification of interaction partners

  • Implement targeted MS approaches for validation of key interactions

• Data analysis considerations:

  • Filter against common contaminants and non-specific interactors

  • Apply statistical methods to identify significantly enriched proteins

  • Validate top candidates with reciprocal IP experiments

  • Perform network analysis to identify functional protein clusters

• Validation of immunity-related interactions:

  • Confirm key interactions by co-immunoprecipitation and Western blot

  • Perform bimolecular fluorescence complementation for in vivo validation

  • Assess functional relevance through genetic interaction studies
    This comprehensive approach can reveal whether AT1G71250 interacts with known immunity components like ATG6 or NPR1 , providing insights into its potential role in plant defense mechanisms.