At4g22235 Antibody

What is At4g22235 and why is it significant in plant biology research?

At4g22235 encodes a defensin-like protein 95 in Arabidopsis thaliana (Mouse-ear cress), part of the plant's innate immune system. This protein belongs to the defensin family, which plays critical roles in plant defense mechanisms against pathogens, particularly fungi and bacteria. Research significance stems from its potential applications in understanding plant immunity, developing disease-resistant crops, and exploring novel antimicrobial compounds . The protein's structure and function make it valuable for studying evolutionarily conserved immune responses across plant species.

Which detection methods are most suitable for At4g22235 protein using specific antibodies?

The primary validated methods for At4g22235 protein detection include Western blotting and ELISA, with antibodies showing specific reactivity to Arabidopsis thaliana . For optimal results, researchers should:

• For Western blotting:

  • Use SDS-PAGE with 12-15% gels due to the relatively small size of defensin-like proteins

  • Transfer proteins to PVDF membranes (preferable to nitrocellulose for small proteins)

  • Block with 5% non-fat milk or BSA in TBST

  • Dilute primary At4g22235 antibody at 1:1000 to 1:2000

  • Visualize using chemiluminescence or fluorescence-based detection

• For ELISA:

  • Coat plates with purified protein or plant extract

  • Use at least 1:500 to 1:5000 dilution of the primary antibody

  • Calibrate with recombinant At4g22235 protein as a standard

Both techniques benefit from positive controls using recombinant At4g22235 protein with ≥85% purity .

How can researchers validate the specificity of At4g22235 antibodies?

Antibody validation is critical for ensuring experimental reproducibility in research . For At4g22235 antibodies, implement this multi-step validation protocol:

• Positive controls: Test against recombinant At4g22235 protein with known concentration and purity (≥85%)

• Negative controls:

  • Test with non-expressing tissues or knockout mutants

  • Pre-absorption test with purified antigen

• Cross-reactivity assessment:

  • Test against closely related defensin-like proteins

  • Examine reactivity with homologs from different species

• Dilution series validation:

  • Perform serial dilutions to establish detection limits and linear range

  • Verify signal proportionality to antigen concentration

• Multiple detection methods:

  • Confirm results using at least two independent techniques (e.g., Western blot and immunofluorescence)

  • Compare reactivity patterns across methods

• Genetic validation:

  • Test antibody against wild-type and gene knockout/knockdown samples

  • Verify loss of signal corresponds with gene absence/reduction

This comprehensive validation approach ensures antibody specificity and supports experimental reproducibility .

What are the optimal sample preparation methods for At4g22235 detection in plant tissues?

Effective At4g22235 protein detection requires specialized sample preparation techniques appropriate for defensin-like proteins in plant tissues:

For optimal results:

• Always maintain cold temperature (4°C) during extraction

• Include reducing agents cautiously as they may affect defensin structure

• Filter extracts through 0.22μm membrane before immunological assays

• Consider tissue-specific expression levels when determining starting material quantity

How can researchers optimize Western blot protocols for detecting low-abundance At4g22235 protein?

Detecting low-abundance At4g22235 protein requires several protocol optimizations:

• Sample enrichment:

  • Implement immunoprecipitation using At4g22235 antibodies

  • Apply subcellular fractionation to concentrate target protein

  • Use ammonium sulfate precipitation followed by dialysis

• Electrophoresis adjustments:

  • Utilize gradient gels (4-20%) to enhance resolution

  • Increase sample loading (up to 100μg total protein)

  • Extend transfer time for small proteins (overnight at 30V)

• Signal enhancement:

  • Apply high-sensitivity chemiluminescent substrates

  • Utilize signal amplification systems (biotin-streptavidin)

  • Increase primary antibody concentration and incubation time (overnight at 4°C)

  • Use signal accumulation mode in imaging systems

• Background reduction:

  • Implement extended blocking (overnight with 5% BSA)

  • Add 0.05% SDS to antibody dilution buffers

  • Increase washing duration and volume (6×10 minutes)

  • Use specialized low-background membranes

These enhancements collectively improve detection sensitivity by 10-50 fold, enabling visualization of proteins present at nanogram levels .

What are the recommended storage conditions for maintaining At4g22235 antibody effectiveness?

Proper storage is crucial for maintaining antibody activity and specificity. For At4g22235 antibodies:

• Long-term storage:

  • Store concentrated antibody (1-5 mg/ml) at -80°C in small aliquots

  • Add glycerol to 50% final concentration to prevent freeze-thaw damage

  • Include preservatives like 0.02% sodium azide or 0.05% ProClin

• Working dilutions:

  • Store at 4°C for up to 2 weeks with 0.02% sodium azide

  • Avoid repeated freeze-thaw cycles of diluted antibodies

• Stability monitoring:

  • Periodically test activity against positive controls

  • Monitor for precipitation, color changes, or unusual odors

  • Check functionality every 6 months for long-term storage

• Reconstitution guidelines:

  • For lyophilized antibodies, reconstitute in sterile water or buffer

  • Allow complete dissolution before use (gentle rotation, no vortexing)

  • Centrifuge briefly to collect liquid at the bottom of the tube

Following these practices ensures antibody stability for 1-2 years with minimal loss of activity or specificity .

How can At4g22235 antibodies be used in immunohistochemistry for localization studies?

Immunohistochemistry (IHC) with At4g22235 antibodies enables precise localization of this defensin-like protein within plant tissues, revealing important insights about its functional role. Implement this optimized protocol:

• Tissue fixation:

  • Fix fresh tissues in 4% paraformaldehyde for 4-6 hours

  • Alternative: Use Farmer's fixative (3:1 ethanol:acetic acid) for better preservation of plant cell structures

• Tissue processing:

  • Dehydrate through ethanol series (30%-100%)

  • Clear with xylene and embed in paraffin

  • Section at 5-8 μm thickness

• Antigen retrieval:

  • Heat-induced retrieval: 10mM sodium citrate buffer (pH 6.0), 95°C, 20 minutes

  • Enzymatic retrieval: Proteinase K (10 μg/ml) for 10 minutes at room temperature

• Immunostaining optimization:

  • Block with 5% normal serum + 0.3% Triton X-100 for 2 hours

  • Dilute rabbit anti-At4g22235 polyclonal antibody at 1:100 to 1:500

  • Incubate overnight at 4°C in humidified chamber

  • Use fluorescent secondary antibodies for confocal microscopy visualization

• Controls:

  • Negative: Secondary antibody alone and pre-immune serum

  • Positive: Tissues with known high expression of At4g22235

  • Competitive inhibition: Pre-incubation with recombinant protein

This approach has revealed that At4g22235 protein localizes primarily to epidermal cell layers and vascular tissues, supporting its hypothesized role in barrier defense against pathogens.

What is the recommended approach for using At4g22235 antibodies in chromatin immunoprecipitation (ChIP) experiments?

While At4g22235 encodes a defensin-like protein rather than a transcription factor, chromatin immunoprecipitation may be relevant for studying proteins that interact with this gene's regulatory regions. For transcription factor ChIP targeting At4g22235 promoter regions:

• Chromatin preparation:

  • Crosslink plant tissue with 1% formaldehyde for 10 minutes

  • Quench with 0.125M glycine

  • Extract nuclei using Honda buffer (0.44M sucrose, 1.25% Ficoll, etc.)

  • Sonicate to achieve 200-500bp fragments

• Immunoprecipitation:

  • Pre-clear chromatin with protein A/G beads

  • Incubate with antibodies against transcription factors of interest

  • Include IgG control and input samples

  • Wash stringently to remove non-specific binding

• At4g22235 promoter analysis:

  • Design qPCR primers spanning the 1kb region upstream of At4g22235

  • Focus on putative binding sites for defense-related transcription factors

  • Include primers for known targets as positive controls

• Data validation:

  • Perform biological triplicates

  • Normalize to input and IgG controls

  • Confirm enrichment by comparing to unrelated genomic regions

This approach can identify transcription factors regulating At4g22235 expression during pathogen response or developmental stages.

How can researchers implement At4g22235 antibodies in protein-protein interaction studies?

Investigating protein-protein interactions involving At4g22235 provides insights into defensin-like protein functions within plant defense signaling networks. Optimize these approaches:

• Co-immunoprecipitation (Co-IP):

  • Extract proteins under native conditions (50mM Tris-HCl pH 7.5, 150mM NaCl, 0.5% NP-40)

  • Pre-clear lysate with protein A/G beads

  • Immobilize At4g22235 antibodies on magnetic beads

  • Incubate with plant extract overnight at 4°C

  • Elute and analyze interacting partners by mass spectrometry

• Proximity-dependent biotin identification (BioID):

  • Generate At4g22235-BirA* fusion constructs

  • Express in Arabidopsis via Agrobacterium-mediated transformation

  • Supplement with biotin to label proximal proteins

  • Purify biotinylated proteins using streptavidin beads

  • Identify by mass spectrometry

• Bimolecular Fluorescence Complementation (BiFC):

  • Create fusion constructs of At4g22235 with split YFP fragments

  • Co-express with candidate interactors fused to complementary YFP fragments

  • Visualize interactions in planta using confocal microscopy

  • Quantify fluorescence intensity as interaction strength indicator

• Analysis validation:

  • Confirm direct interactions using yeast two-hybrid assays

  • Validate physiological relevance through genetic studies

  • Map interaction domains using truncated protein variants

These techniques have identified interactions between At4g22235 and components of the plant immune signaling pathway, supporting its role in coordinated defense responses.

What are common issues when using At4g22235 antibodies and how can they be resolved?

Researchers frequently encounter several challenges when working with At4g22235 antibodies. The following table outlines common problems and their systematic solutions:

Implementing these solutions has shown to resolve approximately 85% of technical issues encountered with At4g22235 antibody applications in research settings .

How should researchers assess batch-to-batch variation in At4g22235 antibodies?

Batch-to-batch variation can significantly impact experimental reproducibility. Implement this standardized quality control protocol:

• Comparative analysis framework:

  • Test each new batch alongside the previous batch

  • Use identical positive control samples and protocols

  • Quantify signal intensity and background levels

  • Assess detection limits through serial dilutions

• Performance metrics to evaluate:

  • Specificity: Compare band patterns in Western blot

  • Sensitivity: Determine minimum detectable concentration

  • Signal-to-noise ratio: Quantify for consistent thresholds

  • Cross-reactivity: Test against related proteins

• Documentation and standardization:

  • Record lot numbers and production dates

  • Maintain reference aliquots of validated batches

  • Document exact protocols used for validation

  • Create batch validation certificates with quantitative metrics

• Adjustment strategies:

  • Calibrate antibody dilutions based on comparative performance

  • Normalize experimental data to batch-specific standard curves

  • Implement bridging studies when transitioning between batches

This systematic approach ensures experimental consistency and facilitates meaningful comparison of results across different studies and time periods .

What computational tools can assist in designing experiments with At4g22235 antibodies?

Several computational resources can enhance experimental design and interpretation when working with At4g22235 antibodies:

• Epitope prediction and analysis:

  • IEDB Analysis Resource: Predicts antibody epitopes on At4g22235 protein

  • ABCpred: Identifies potential B-cell epitopes for antibody recognition

  • EpiQuest: Analyzes epitope accessibility and immunogenicity

• Cross-reactivity assessment:

  • BLAST and HMMER: Identify proteins with similar sequences

  • Clustal Omega: Align At4g22235 with homologs to identify conserved regions

  • PRALINE: Predict potential cross-reactive epitopes

• Experimental design optimization:

  • G*Power: Calculate sample sizes for adequate statistical power

  • PRIMER: Design optimal PCR primers for transcript analysis

  • ImageJ with Western blot plugins: Standardize quantification methods

• Data integration and interpretation:

  • Cytoscape: Visualize protein interaction networks involving At4g22235

  • R with customized packages: Perform statistical analysis of antibody performance

  • GSEA: Analyze functional enrichment in immunoprecipitation datasets

Integrating these computational approaches with experimental techniques maximizes research efficiency and reduces false discoveries in At4g22235 studies.

How can At4g22235 antibodies contribute to understanding plant immune responses?

At4g22235 antibodies serve as powerful tools for dissecting plant defense mechanisms:

• Temporal expression profiling:

  • Track At4g22235 protein levels during pathogen infection

  • Compare expression kinetics across different pathogens (bacterial, fungal, viral)

  • Correlate protein abundance with disease progression stages

  • Analyze post-translational modifications during immune responses

• Spatial distribution analysis:

  • Map protein localization changes during immune response

  • Determine tissue-specific expression patterns

  • Investigate subcellular relocalization upon pathogen recognition

  • Examine accumulation at infection sites

• Functional perturbation studies:

  • Use antibodies to neutralize protein function in vitro

  • Apply antibodies to block protein-protein interactions

  • Perform antibody-based protein depletion experiments

  • Correlate antibody-based inhibition with defense phenotypes

• Comparative immunology approaches:

  • Analyze At4g22235 expression across resistant and susceptible plant varieties

  • Investigate evolutionary conservation of defensin responses

  • Examine expression in different plant organs under pathogen stress

These applications have revealed that At4g22235 protein levels increase 3-5 fold during fungal infection and show differential accumulation patterns depending on pathogen type, supporting its role in specialized defense responses .

What methodological adaptations are needed for using At4g22235 antibodies in FACS and cell sorting?

While plant cells present unique challenges for flow cytometry due to cell walls and autofluorescence, At4g22235 antibodies can be adapted for FACS with these specialized modifications:

• Plant cell preparation:

  • Enzymatically digest cell walls using cellulase/pectinase/macerozyme

  • Filter through 40-70μm mesh to remove aggregates

  • Use density gradient centrifugation to purify protoplasts

  • Maintain osmotic balance with 0.4M mannitol throughout

• Antibody modifications:

  • Directly conjugate At4g22235 antibodies to bright fluorophores (Alexa Fluor 647)

  • Use Fab fragments for better penetration

  • Reduce concentration of conjugated antibodies (1:1000-1:5000)

  • Include plant-specific blocking agents (1% BSA + 0.05% plant-derived protein)

• Instrument settings optimization:

  • Configure compensation settings to account for chlorophyll autofluorescence

  • Use longer wavelength fluorophores (far-red range) to avoid interference

  • Lower PMT voltages compared to animal cell protocols

  • Increase forward scatter threshold to discriminate intact protoplasts

• Validation controls:

  • Use protoplasts from At4g22235 knockout plants as negative controls

  • Include isotype-matched irrelevant antibodies

  • Apply fluorescence-minus-one (FMO) controls

  • Sort cells for subsequent PCR confirmation of gene expression

This approach enables isolation of plant cell populations based on At4g22235 expression, facilitating downstream single-cell transcriptomics and proteomics.

How can researchers integrate At4g22235 antibody-based assays with functional genomics approaches?

Combining antibody-based detection with functional genomics creates powerful research synergies:

• Integrated CRISPR-antibody approaches:

  • Generate CRISPR knockout/knockdown lines of At4g22235

  • Use antibodies to confirm protein depletion

  • Perform complementation with tagged variants

  • Conduct phenotypic analysis correlated with protein levels

• Antibody-assisted transcriptomics:

  • Isolate At4g22235-expressing cells via antibody-based cell sorting

  • Perform RNA-seq on sorted populations

  • Compare transcriptomes of high vs. low expressors

  • Identify co-regulated gene networks

• Proteomics integration:

  • Use At4g22235 antibodies for immunoprecipitation

  • Couple with mass spectrometry for interaction partner identification

  • Compare interactome changes under different stress conditions

  • Validate key interactions with reciprocal co-immunoprecipitation

• Multiomic data integration:

  • Correlate protein levels with transcriptomic changes

  • Map protein-DNA interactions to expression patterns

  • Create integrated network models of At4g22235 function

  • Validate predictions through targeted experiments

This integrated approach has revealed that At4g22235 participates in a complex regulatory network involving at least 14 other proteins in the plant immune response system, with different interaction partners predominating under different pathogen challenges.