At3g27835 Antibody

What is the At3g27835 gene and what protein does it encode?

At3g27835 is a gene locus in Arabidopsis thaliana that encodes a protein involved in plant immune responses. Understanding this protein's function is essential for researchers studying plant-pathogen interactions. While specific information about At3g27835 is limited in the current literature, it likely functions similarly to other small GTPases like AtRAC7/ROP9, which regulate immune responses in Arabidopsis against pathogens such as Botrytis cinerea and Pseudomonas syringae . Methodologically, researchers should consider gene expression analysis through RT-PCR or RNA-seq to confirm expression patterns before developing antibodies against the encoded protein.

How are antibodies against plant proteins like At3g27835 typically generated?

Antibodies against plant proteins are commonly generated through several approaches:

• Monoclonal antibody production: Similar to the LM18, LM19, and LM20 antibodies developed for Arabidopsis seed coat mucilage, researchers isolate and screen for binding specificity to the target protein . This involves:

  • Recombinant expression of the target protein or protein fragment

  • Immunization of rats or mice with the purified protein

  • Hybridoma cell generation and clonal selection

  • Validation through binding assays against both recombinant protein and plant tissue samples

• Computational design approaches: Recent advancements leverage active learning and AI-accelerated approaches to improve antibody development, particularly for proteins with challenging binding properties .

What are the recommended storage conditions for At3g27835 antibodies?

Plant protein antibodies like those targeting At3g27835 require specific storage conditions to maintain functionality:

• Store at -20°C for long-term storage

• Avoid repeated freeze-thaw cycles (aliquot upon receipt)

• Include stabilizing proteins such as BSA (0.1-1%)

• Store working dilutions at 4°C for no more than one week

• Include sodium azide (0.02%) for preservative purposes if not used for functional assays

These recommendations follow standard practices for maintaining antibody integrity and preventing degradation or aggregation that could compromise experimental results.

What immunoblotting techniques are most effective for At3g27835 antibody?

For optimal immunoblotting with plant protein antibodies like those targeting At3g27835:

• Sample preparation:

  • Extract total protein from plant tissue using buffer containing:

    • 50 mM Tris-HCl (pH 7.5)

    • 150 mM NaCl

    • 1% Triton X-100

    • Protease inhibitor cocktail

  • Use fresh tissue whenever possible or flash-freeze in liquid nitrogen

• Gel electrophoresis and transfer:

  • 10-12% SDS-PAGE gels typically work well for most plant proteins

  • Transfer to PVDF membranes (preferred over nitrocellulose for plant samples)

  • Use wet transfer systems at lower voltage (30V) overnight at 4°C to improve transfer efficiency

• Blocking and antibody incubation:

  • Block with 5% non-fat dry milk in TBST (plant samples often have higher background)

  • Primary antibody dilution: Start with 1:1000 and optimize

  • Include negative controls (pre-immune serum) and positive controls (recombinant protein)

• Detection:

  • HRP-conjugated secondary antibodies with ECL detection systems

  • Consider signal enhancement systems for low-abundance proteins

How can I optimize immunoprecipitation protocols for At3g27835 protein studies?

Optimizing immunoprecipitation for plant proteins requires special considerations:

• Buffer optimization:

  Buffer Component	Concentration	Purpose
  Tris-HCl (pH 7.5)	50 mM	Maintains pH
  NaCl	150 mM	Ionic strength
  EDTA	1 mM	Chelates divalent cations
  Triton X-100	0.5%	Membrane solubilization
  Glycerol	5%	Stabilizes proteins
  Protease inhibitors	1X	Prevents degradation
  Phosphatase inhibitors	1X	Preserves phosphorylation

• Pre-clearing strategy:

  • Pre-clear lysates with Protein A/G beads and pre-immune serum

  • Extend pre-clearing time to 2 hours (plant extracts require longer pre-clearing)

• Antibody coupling:

  • Covalently couple antibodies to beads when possible

  • Use crosslinkers that don't interfere with antibody-antigen binding

• Elution conditions:

  • Test both low pH and competitive elution methods

  • Optimize elution conditions to maintain protein integrity and activity

What considerations are important for performing immunohistochemistry with At3g27835 antibodies?

For effective immunohistochemistry in plant tissues:

• Fixation and embedding:

  • 4% paraformaldehyde fixation (12-24 hours)

  • Gradual dehydration series (30%, 50%, 70%, 90%, 100% ethanol)

  • Paraffin embedding with extended infiltration times compared to animal tissues

• Antigen retrieval:

  • Critical for plant tissues due to cell wall interference

  • Citrate buffer (pH 6.0) heating or enzymatic methods

  • Consider cell wall-degrading enzymes (cellulase, pectinase) for improved antibody access

• Antibody incubation:

  • Longer incubation times (overnight at 4°C)

  • Higher antibody concentrations may be required

  • Include appropriate controls, similar to those used for monoclonal antibodies to pectic homogalacturonan

• Detection:

  • Fluorescent secondary antibodies with confocal microscopy

  • Autofluorescence control sections essential for plant tissues

How can active learning approaches improve antibody development for plant proteins like At3g27835?

Active learning methodologies can significantly enhance antibody development for challenging plant targets:

• Library-on-library screening optimization:

  • Using machine learning models to predict antibody-antigen binding

  • Creating mutant libraries with strategic variation

  • Iterative experimental validation focusing on high-confidence predictions

• Performance improvement metrics:

  • Recent studies showed up to 35% reduction in required antigen mutant variants

  • Learning process acceleration by approximately 28 steps compared to random approaches

  • Improved out-of-distribution prediction performance

• Practical implementation strategy:

  • Begin with small labeled dataset

  • Apply predictive algorithms to select highest-information variants for testing

  • Iteratively expand labeled dataset based on model uncertainty

  • Validate binding predictions with experimental confirmation

This approach is particularly valuable for plant proteins with limited prior characterization, such as At3g27835.

What are the latest approaches for reducing antibody self-interaction for plant protein studies?

Self-interaction of antibodies can compromise experimental results. Advanced approaches include:

• Computational analysis:

  • Spatial aggregation propensity (SAP) assessment of antibody structure

  • Solvent-accessible surface area (SASA) analysis

  • Combined SAP/SASA approach to identify self-interaction hotspots

• Strategic mutagenesis:

  • Target residues with high SASA (>50Ų) and high SAP score (>2.3)

  • Secondary targets: residues with high SASA (>50Ų) and moderate SAP (1.0-2.3)

  • Tertiary targets: moderate SASA (20-50Ų) and high SAP (>2.3)

• Screening methodology:

  • Dynamic light scattering (DLS) as a medium-throughput initial screen

  • Measure interaction parameters at low concentration (2-10 mg/mL)

  • Validate promising variants with viscosity measurements at high concentration

• Performance metrics for optimized antibodies:

  Modification	Viscosity Improvement	Interaction Parameter	Antigen Binding
  CDR modifications	Up to 8-fold	Significantly improved	Within few-fold of parent
  Surface hydrophobicity reduction	2-4 fold	2-3 fold improvement	Minimally affected

How do translational barriers impact antibody-based studies of plant proteins?

Several translational barriers affect antibody-based studies of plant proteins:

• Accessibility barriers:

  • Cell wall presents physical barrier for in vivo applications

  • Specialized delivery mechanisms needed for intracellular targets

  • Consider polymer-based modifications similar to those used for therapeutic antibodies crossing the blood-brain barrier

• Specificity challenges:

  • High homology between plant protein families

  • Cross-reactivity with related proteins can complicate interpretation

  • Extensive validation against knockout/mutant lines essential

• Expression system considerations:

  • Prokaryotic vs. eukaryotic expression systems impact post-translational modifications

  • Plant-specific glycosylation patterns may affect antibody recognition

  • Use of plant-based expression systems for recombinant antibody production

What are the most common causes of false positives in At3g27835 antibody experiments?

False positives in plant antibody experiments commonly result from:

• Cross-reactivity issues:

  • Antibodies may recognize related plant proteins with similar epitopes

  • Test antibody against multiple plant tissues and species

  • Include knockout/mutant lines as negative controls

• Non-specific binding:

  • Plant tissues contain compounds that can non-specifically bind antibodies

  • Optimize blocking conditions with different agents (milk, BSA, plant-derived blockers)

  • Increase washing stringency and duration

• Autofluorescence interference:

  • Plant tissues exhibit significant autofluorescence

  • Include unstained controls and spectral unmixing in microscopy

  • Consider alternative detection methods or fluorophores

• Data validation approach:

  • Always confirm antibody results with complementary techniques (RNA expression, GFP fusion proteins)

  • Include epitope-tagged protein expressions as positive controls

  • Validate with multiple antibodies targeting different regions of the protein

How can researchers verify the specificity of new At3g27835 antibodies?

Comprehensive antibody validation requires multiple approaches:

• Genetic validation:

  • Test against knockout/knockdown lines

  • Overexpression lines should show increased signal

  • CRISPR-generated epitope modifications

• Biochemical validation:

  • Peptide competition assays to confirm epitope specificity

  • Western blot band pattern analysis

  • Mass spectrometry confirmation of immunoprecipitated proteins

• Orthogonal validation:

  • Multiple antibodies targeting different epitopes

  • Correlation with RNA expression data

  • Comparison with GFP-fusion protein localization

• Cross-reactivity assessment:

  • Testing against closely related proteins

  • Multi-species testing to evaluate conservation

  • Testing in different tissue types

What techniques can help resolve conflicting data from antibody-based experiments?

When faced with conflicting antibody data:

• Technical reconciliation approaches:

  • Standardize sample preparation methods

  • Implement quantitative controls in each experiment

  • Evaluate antibody lot-to-lot variation

• Alternative detection methods:

  • RNA-based expression analysis (RNA-seq, qRT-PCR)

  • Activity-based protein profiling

  • Genetic reporters (GFP/YFP fusions)

• Protein characteristic analysis:

  • Post-translational modification assessment

  • Protein complex formation evaluation

  • Subcellular localization studies

• Advanced validation strategy:

  • Proximity labeling approaches

  • Single-cell analysis to assess heterogeneity

  • Developing complementary methodologies specific to plant systems, similar to approaches used in monoclonal antibody research for pectic homogalacturonan