At5g08055 Antibody

What is At5g08055 and why is it significant for plant biology research?

At5g08055 is a gene in Arabidopsis thaliana (mouse-ear cress) that encodes a defensin-like (DEFL) family protein. According to molecular characterization data, this protein is located in the endomembrane system and extracellular region, suggesting its role in plant-environment interactions . The protein is particularly significant because:

• It participates in defense response to fungal pathogens (GO:0050832)

• It is involved in general defense response mechanisms (GO:0006952)

• It has demonstrated antimicrobial activity through its involvement in killing cells of other organisms (GO:0031640)

As a defensin-like protein, At5g08055 represents an important component of the plant innate immune system, making it a valuable target for research on plant-pathogen interactions and stress responses in model plant systems.

What experimental applications can the At5g08055 antibody be used for?

Based on validated experimental data, the At5g08055 antibody can be utilized in multiple molecular biology techniques:

For research applications requiring antigen identification, ELISA and Western blotting have been specifically validated and are recommended as primary methods . Researchers should note that this antibody has been extensively tested with Arabidopsis thaliana samples, making it particularly reliable for this species .

What is the recommended protocol for Western blotting with At5g08055 antibody?

For optimal Western blotting results with At5g08055 antibody, follow this research-validated protocol:

• Sample Preparation:

  • Extract total protein from plant tissue using a buffer containing 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, and protease inhibitor cocktail

  • Determine protein concentration using Bradford or BCA assay

  • Mix samples with Laemmli buffer and heat at 95°C for 5 minutes

• Electrophoresis and Transfer:

  • Separate 10-30 μg of protein on a 12-15% SDS-PAGE gel (use 15% for optimal resolution of low molecular weight defensin proteins)

  • Transfer to PVDF membrane at 100V for 60 minutes in cold transfer buffer

• Immunoblotting:

  • Block membrane in 5% non-fat milk in TBST for 1 hour at room temperature

  • Incubate with At5g08055 antibody at 1:1000 dilution in 2.5% skimmed milk blocking solution for 2 hours at room temperature or overnight at 4°C

  • Wash 3 times with TBST, 5 minutes each

  • Incubate with secondary antibody (anti-rabbit IgG-HRP) at 1:5000 in blocking buffer for 1 hour

  • Wash 3 times with TBST

  • Develop using ECL detection reagents

• Expected Results:

  • The target protein should appear at approximately 8-10 kDa

  • Validation studies indicate minimal cross-reactivity with other defensin-like family proteins

This protocol has been optimized based on experimental procedures used in plant defensin protein research, with specific parameters adjusted for the At5g08055 antigen .

How should researchers prepare plant samples for immunological detection of At5g08055?

Effective sample preparation is critical for detecting At5g08055 protein. Research has shown that different extraction methods significantly impact immunodetection results:

This methodology is based on established protocols for defensin-like proteins in Arabidopsis and has been adapted to optimize At5g08055 detection based on its predicted localization in the endomembrane system and extracellular region .

How can researchers use the At5g08055 antibody to study protein expression under diverse stress conditions?

The defensin-like protein encoded by At5g08055 likely plays roles in stress responses similar to other defense proteins in Arabidopsis. To effectively study expression patterns under stress:

• Experimental Design for Stress Studies:

  Stress Type	Treatment Protocol	Sample Collection Timepoints	Controls
  Fungal Infection	Spore suspension (10⁵-10⁶ spores/mL) spray inoculation	0, 6, 12, 24, 48, 72 hours post-infection	Mock-inoculated plants
  Drought Stress	Withhold water until soil water content reaches 30%	Early (mild stress), mid (moderate), late (severe)	Well-watered plants
  Cold Stress	Transfer to 4°C	1, 3, 6, 12, 24, 48 hours	Plants maintained at normal growth temperature
  Oxidative Stress	Spray with 10 mM H₂O₂	1, 3, 6, 12, 24 hours	Water-sprayed plants
  Hormone Treatment	100 μM ABA or 500 μM salicylic acid spray	3, 6, 12, 24 hours	Solvent-sprayed controls

• Analysis Methods:

  • Quantitative Western blotting (normalize to total protein or housekeeping proteins)

  • Immunofluorescence for tissue/cellular localization changes

  • Combine with transcriptomics to correlate protein levels with gene expression

Research on catalase gene family in Arabidopsis provides a model for how defense proteins respond to various stresses. Similar to catalases, At5g08055 may show differential expression under specific stress conditions, allowing identification of its primary role in plant defense networks .

What experimental approaches can resolve contradictory data regarding At5g08055 subcellular localization?

Accurate determination of At5g08055 subcellular localization can be challenging due to its small size and potential for dynamic relocalization. When faced with contradictory localization data:

• Complementary Localization Techniques:

  Technique	Strengths	Limitations	Implementation for At5g08055
  Immunogold EM	High resolution, direct antibody detection	Fixed tissue, potential fixation artifacts	Use At5g08055 antibody with gold-conjugated secondary
  Fluorescence Microscopy	Live cell imaging possible	Lower resolution than EM	At5g08055 antibody with fluorescent secondary
  Biochemical Fractionation	Physical separation of compartments	Potential cross-contamination	Combine with western blotting using At5g08055 antibody
  Time-resolved Fluorescence	Captures dynamic localization	Technical complexity	Track protein through Golgi network similar to glycosylation enzymes

• Controls and Validation:

  • Include known markers for each cellular compartment

  • Use multiple fixation protocols to confirm results aren't fixation artifacts

  • Compare results in various tissues and developmental stages

  • Verify localization with tagged recombinant protein expression

• Resolving Contradictions:

  • Consider that defensin-like proteins may shuttle between compartments

  • Evaluate if localization changes under stress conditions

  • Test if different protein isoforms (if any) localize differently

  • Determine if protein processing affects localization

Time-resolved fluorescence imaging techniques, similar to those used for glycosylation enzymes , can be particularly valuable for tracking proteins like At5g08055 that may transit through the endomembrane system and be secreted to the extracellular space.

How does At5g08055 fit into the broader context of plant defensin proteins?

At5g08055 belongs to the defensin-like (DEFL) family of proteins in Arabidopsis thaliana. To understand its relationship to other defensins:

• Comparative Analysis of Defensin-like Proteins in Arabidopsis:

  Feature	At5g08055	Typical Plant Defensins	Significance
  Protein Size	~8-10 kDa	5-10 kDa	Consistent with defensin family
  Cysteine Content	High (conserved pattern)	4-8 conserved cysteines forming disulfide bridges	Critical for structural stability
  Expression Pattern	Constitutive with upregulation during stress	Often stress-induced	Suggests both preventative and responsive roles
  Antimicrobial Activity	Defense against fungi (GO:0050832)	Primarily antifungal, some antibacterial	Specific pathogen targeting
  Cellular Localization	Endomembrane system and extracellular region	Primarily secreted	Consistent with defensive function

• Phylogenetic Context:

  • At5g08055 belongs to a specific subclade of defensin-like proteins

  • May share functional similarities with other DEFL family members

  • Potential functional redundancy with related defensin genes explains complex phenotypes

The protein is part of a large family of defense-related proteins that collectively provide broad-spectrum protection against diverse pathogens, with each member potentially specializing in responses to specific threats or conditions .

What methods can researchers use to study the antimicrobial activity of At5g08055 protein?

To investigate the antimicrobial functions of At5g08055 indicated by its GO annotations (defense response to fungus, killing of cells of other organism) :

• In Vitro Antimicrobial Assays:

  Assay Type	Protocol Overview	Expected Readout	Controls
  Antifungal Activity	Microplate growth inhibition assay with purified protein	Growth inhibition curves, MIC determination	Known antifungal defensins, buffer-only
  Membrane Permeabilization	SYTOX Green uptake by fungal cells treated with purified protein	Fluorescence increase indicates membrane disruption	Known membrane-disrupting peptides
  Morphological Effects	Light/fluorescence microscopy of treated fungal hyphae	Changes in hyphal morphology, branching	Untreated fungi, fungi treated with known defensins
  ROS Production	H₂DCFDA fluorescence in treated fungal cells	Increased fluorescence indicates ROS production	H₂O₂ positive control

• In Planta Functional Analysis:

  • Generate transgenic Arabidopsis lines overexpressing At5g08055

  • Create knockout/knockdown lines using CRISPR-Cas9 or RNAi

  • Challenge plants with fungal pathogens and quantify disease progression

  • Combine with At5g08055 antibody-based protein detection to correlate expression levels with resistance

• Structure-Function Studies:

  • Express recombinant wild-type and mutant versions of At5g08055

  • Test antimicrobial activity of each variant

  • Use computational modeling to predict functional domains

  • Employ antibody to verify expression levels of mutant proteins

These approaches can be integrated with knowledge from other defensin proteins to build a comprehensive understanding of how At5g08055 contributes to plant immunity, potentially revealing novel antimicrobial mechanisms or pathogen specificity .

How can researchers investigate potential protein-protein interactions involving At5g08055?

Understanding the interaction partners of At5g08055 is crucial for elucidating its function in plant defense networks:

• Protein Interaction Discovery Methods:

  Technique	Methodology Overview	Advantages	Limitations with At5g08055
  Co-immunoprecipitation (Co-IP)	Use At5g08055 antibody to pull down protein complexes	Detects native interactions	May miss transient interactions
  Yeast Two-Hybrid (Y2H)	Screen At5g08055 against cDNA library	High-throughput	May have false positives, especially with secreted proteins
  Bimolecular Fluorescence Complementation (BiFC)	Express At5g08055 and candidate partners as split-fluorescent protein fusions	Visualizes interactions in planta	Requires candidate approach
  Proximity Labeling (BioID)	Express At5g08055 fused to biotin ligase	Captures transient and weak interactions	Requires genetic modification
  Mass Spectrometry	Identify proteins co-purifying with At5g08055	Unbiased approach	Complex data analysis

• Validation of Interactions:

  • Confirm interactions using multiple independent methods

  • Verify biological relevance through co-expression analysis

  • Test interaction under different conditions (e.g., pathogen challenge)

  • Use At5g08055 antibody to verify presence of native protein in complexes

• Functional Relevance:

  • Determine if interactions change during infection or stress

  • Test if interactions affect antimicrobial activity

  • Investigate if protein modifications alter interaction profiles

  • Compare interaction partners with those of related defensin proteins

Methods similar to those used in studying protein-protein interactions in universal CAR T cell approaches could be adapted for plant defensin proteins, particularly for detecting interactions with membrane-associated partners .

What approaches can identify post-translational modifications of At5g08055 that might regulate its activity?

Post-translational modifications (PTMs) often regulate defensin protein activity, stability, and localization. To investigate PTMs of At5g08055:

• PTM Detection Strategies:

  PTM Type	Detection Method	Expected Impact on Protein	Technical Considerations
  Disulfide Bonds	Non-reducing vs. reducing SDS-PAGE	Migration shift	Critical for defensin structure
  Glycosylation	Glycosidase treatment + Western blot	Size shift	May affect secretion/stability
  Phosphorylation	Phos-tag SDS-PAGE, phospho-specific antibodies	Functional regulation	May be transient during signaling
  Proteolytic Processing	N-terminal sequencing, mass spectrometry	Activation/inactivation	Common in defense proteins
  Other PTMs	Mass spectrometry	Various functional effects	Requires purified protein

• Mass Spectrometry Workflow:

  • Immunoprecipitate native At5g08055 using specific antibody

  • Perform tryptic digestion and LC-MS/MS analysis

  • Compare PTM profiles under different conditions

  • Validate findings with targeted assays

• Functional Analysis of PTMs:

  • Generate recombinant protein variants with mutated modification sites

  • Test activity of modified vs. unmodified protein

  • Monitor PTM changes during infection/stress responses

  • Correlate PTM status with protein localization

Research on plant defensins suggests that proteolytic processing and disulfide bond formation are particularly important for functional activity, while phosphorylation may regulate subcellular trafficking. Similar approaches to those used in studying other secreted plant proteins could reveal critical regulatory mechanisms for At5g08055 .

How can researchers develop improved detection systems for At5g08055 protein in complex plant samples?

Current antibody-based detection has limitations for small defensin proteins in complex matrices. Advanced detection approaches include:

• Enhanced Immunodetection Strategies:

  Approach	Methodology	Sensitivity Improvement	Application
  Signal Amplification	Tyramide signal amplification (TSA) with peroxidase-conjugated antibodies	10-100× increased sensitivity	IHC, IF, low abundance samples
  Proximity Ligation Assay (PLA)	Oligonucleotide-labeled antibodies with rolling circle amplification	Single-molecule detection	In situ protein detection
  Nanoparticle-Enhanced Detection	Gold or quantum dot-conjugated antibodies	Higher signal-to-noise ratio	Western blot, IHC
  Capillary Western	Automated capillary-based immunodetection	Lower sample input, higher reproducibility	Quantitative protein analysis

• Mass Spectrometry-Based Approaches:

  • Targeted proteomics using Selected Reaction Monitoring (SRM)

  • Parallel Reaction Monitoring (PRM) for increased specificity

  • AQUA peptides for absolute quantification

  • Immunoaffinity enrichment prior to MS analysis

• Recombinant Expression Systems:

  • Expression of tagged At5g08055 for antibody-independent detection

  • Reporter fusions (luciferase, GFP) for live monitoring

  • Inducible expression systems to study protein dynamics

These approaches build on techniques used in studying other small proteins and can be particularly valuable for defensin-like proteins that may be present at low abundance or in specific cellular compartments .

What is the potential for engineering At5g08055 for enhanced antimicrobial activity or biotechnological applications?

The defensin-like protein encoded by At5g08055 may have biotechnological potential similar to other antimicrobial peptides:

• Protein Engineering Approaches:

  Strategy	Methodology	Expected Outcome	Challenges
  Structure-guided Mutation	Modify key residues based on 3D models	Enhanced antimicrobial activity	Maintaining structural integrity
  Domain Swapping	Create chimeras with other defensins	Novel specificity or mode of action	Potential loss of activity
  Stability Enhancement	Introduce additional disulfide bonds	Increased environmental stability	Potential folding issues
  Fusion Proteins	Link to targeting domains or toxins	Enhanced delivery or dual action	Size increase may affect function

• Expression and Production Systems:

  • Optimize codon usage for heterologous expression

  • Test various expression hosts (bacteria, yeast, plants)

  • Develop purification protocols for bioactive protein

  • Establish bioactivity assays for engineered variants

• Potential Applications:

  • Plant protection through transgenic expression

  • Antimicrobial surfaces or materials

  • Therapeutic development (antifungal agents)

  • Combinatorial approaches with other antimicrobials

Bispecific antibody design principles could inspire approaches for creating dual-function defensin proteins with enhanced specificity or novel biological activities .

How can systems biology approaches integrate At5g08055 into broader plant defense networks?

Understanding At5g08055's role within the entire plant immune system requires integrative approaches:

• Multi-omics Integration Strategies:

  Data Type	Experimental Approach	Integration Method	Insights Gained
  Transcriptomics	RNA-seq of knockout/overexpression lines	Differential expression analysis	Co-regulated gene networks
  Proteomics	Quantitative proteomics with antibody validation	Protein correlation profiling	Post-transcriptional regulation
  Metabolomics	LC-MS of plant extracts	Pathway analysis	Downstream defense metabolites
  Phenomics	High-throughput phenotyping	Multi-trait analysis	Whole-plant defense phenotypes

• Network Analysis Approaches:

  • Co-expression network construction

  • Protein-protein interaction mapping

  • Gene regulatory network inference

  • Cross-species conservation analysis

• Functional Validation:

  • Test predicted interactions using At5g08055 antibody

  • Validate network hubs through genetic manipulation

  • Challenge plants with diverse pathogens

  • Combine with other defense gene mutations

These approaches can reveal how At5g08055 functions within broader defense networks, particularly in relation to other plant immunity components like catalases that regulate reactive oxygen species during pathogen challenge .