At4g22230 Antibody

What is the At4g22230 Antibody and what protein does it specifically target?

The At4g22230 antibody is a research reagent that specifically recognizes and binds to the defensin-like (DEFL) family protein encoded by the AT4G22230 gene in Arabidopsis thaliana. This protein belongs to a class of small, cysteine-rich peptides that play crucial roles in plant defense mechanisms, antimicrobial activity, and developmental regulation. The antibody enables researchers to investigate the protein's expression patterns, subcellular localization, and functional roles within plant biological systems.

When using this antibody, researchers should be aware that:

• It is formulated in a buffer containing 50% glycerol, 0.01M phosphate buffered saline (PBS), and 0.03% Proclin 300 as a preservative

• Its specificity has been validated for Arabidopsis thaliana, though cross-reactivity with orthologous proteins in other plant species requires independent verification

What experimental applications is the At4g22230 Antibody suitable for?

The At4g22230 antibody facilitates multiple experimental applications in plant molecular biology research:

• Immunoblotting (Western blot): For detecting and quantifying At4g22230 protein expression levels in plant tissue extracts

• Immunohistochemistry/Immunofluorescence: For visualizing the spatial distribution of At4g22230 protein in plant tissues

• Immunoprecipitation: For isolating At4g22230 protein complexes to identify interaction partners

• ELISA: For quantitative measurement of protein levels

Optimization recommendations for these applications include:

• Titration of antibody concentration due to variable expression levels of DEFL proteins under different conditions

• Inclusion of appropriate positive and negative controls to validate specificity

• Consideration of tissue-specific expression patterns when designing experiments

What methodological approaches can optimize At4g22230 Antibody specificity validation in non-model plant species?

When validating At4g22230 antibody specificity in non-model plant species, researchers should implement a multi-step approach:

• Sequence homology analysis: Compare the amino acid sequence of At4g22230 with putative orthologs in the target species to predict potential cross-reactivity

• Recombinant protein controls: Express the orthologous protein from the target species and use it as a positive control

• Knockout/knockdown validation: If available, use genetic knockouts or RNAi lines of the orthologous gene to confirm specificity

• Pre-absorption tests: Pre-incubate the antibody with purified recombinant antigen before immunodetection to confirm binding specificity

• Peptide competition assays: Perform parallel detections with and without competing peptide to verify signal specificity

This methodological approach is particularly important given that the At4g22230 antibody has been specifically validated for Arabidopsis thaliana, and cross-reactivity with other species requires verification.

How can researchers integrate At4g22230 Antibody with transcriptomic approaches to understand gene function?

Integrating At4g22230 antibody-based protein detection with transcriptomic data provides a powerful approach to understanding gene function:

• Correlation analysis between protein and mRNA levels:

  • Measure At4g22230 protein levels using the antibody in immunoblotting or ELISA

  • Parallel RNA-seq or qRT-PCR analysis of AT4G22230 gene expression

  • Compare protein abundance with transcript levels to identify post-transcriptional regulation

• Time-course experiments during stress responses:

  • Follow the approach used in pattern-triggered immunity studies where time-resolved transcriptomics revealed dynamic gene expression patterns

  • Compare transcript induction timing with protein accumulation

• Cell-type specific expression analysis:

  • Combine immunolocalization using the At4g22230 antibody with cell-type-specific transcriptomics

• Co-expression network analysis:

  • Identify genes co-expressed with AT4G22230 using transcriptomic data

  • Validate protein-level interactions using co-immunoprecipitation with the At4g22230 antibody

This integrated approach enables researchers to distinguish between transcriptional and post-transcriptional regulation mechanisms affecting At4g22230 protein function.

What experimental design best addresses variation in At4g22230 expression under different stress conditions?

To effectively study At4g22230 expression variation under different stress conditions, researchers should consider this experimental design:

• Stress treatments setup:

  • Apply multiple stress conditions (pathogen infection, drought, salt, cold, heat)

  • Include time-course sampling (e.g., 0, 1, 3, 6, 12, 24 hours)

  • Maintain appropriate biological replicates (minimum n=3)

• Multi-level analysis:

  Analysis Level	Technique	Purpose
  Transcriptional	RT-qPCR	Quantify mRNA expression changes
  Protein	Western blot with At4g22230 antibody	Measure protein accumulation
  Spatial	Immunohistochemistry	Determine tissue-specific localization
  Functional	Phenotypic assessment of mutants	Evaluate physiological significance

• Controls and normalization:

  • Include housekeeping genes/proteins unaffected by the stresses

  • Consider general stress markers to distinguish general vs. specific responses

  • Include pattern-triggered immunity markers for comparison with defense responses

• Statistical analysis:

  • Apply appropriate statistical methods to identify significant changes

  • Consider multiple testing corrections when analyzing across conditions

This approach is informed by methodologies used in transcriptional landscape studies of pattern-triggered immunity in Arabidopsis, where time-resolved sampling revealed distinct expression patterns in response to different stimuli .

How can At4g22230 Antibody be used to investigate protein-protein interactions in the context of plant immunity?

To investigate protein-protein interactions involving At4g22230 in plant immunity contexts:

• Co-immunoprecipitation (Co-IP):

  • Use At4g22230 antibody to pull down the protein complex from plant tissue extracts

  • Identify interaction partners using mass spectrometry

  • Verify interactions with reciprocal Co-IP using antibodies against putative partners

• Proximity labeling techniques:

  • Create fusion proteins with BioID or APEX2 proximity labeling enzymes

  • Use the At4g22230 antibody to confirm expression and localization of fusion proteins

  • Identify proximal proteins through biotinylation and subsequent purification

• Bimolecular Fluorescence Complementation (BiFC):

  • Generate fusion constructs with split fluorescent protein fragments

  • Use At4g22230 antibody in parallel experiments to confirm expression levels

  • Visualize interactions through reconstituted fluorescence

• FRET/FLIM analysis:

  • Create fluorescent protein fusions with At4g22230 and candidate interactors

  • Use the At4g22230 antibody to validate expression of fusion proteins

  • Measure energy transfer to detect direct interactions

This approach aligns with research methodologies examining protein functions in immune signaling pathways, where protein interactions are crucial for signal transduction in pattern-triggered immunity responses .

What sample preparation protocols optimize At4g22230 protein detection in plant tissues?

For optimal detection of At4g22230 protein in plant tissues, the following sample preparation protocol is recommended:

• Tissue collection and processing:

  • Harvest plant tissue at appropriate developmental stage (consider expression timing)

  • Flash-freeze in liquid nitrogen to preserve protein integrity

  • Grind tissue to fine powder while maintaining frozen state

• Protein extraction buffer optimization:

  Component	Concentration	Purpose
  Tris-HCl pH 7.5	50 mM	Buffering
  NaCl	150 mM	Ionic strength
  EDTA	1 mM	Inhibits metalloproteases
  Triton X-100 or NP-40	0.5-1%	Membrane solubilization
  Protease inhibitor cocktail	1×	Prevents degradation
  DTT or β-mercaptoethanol	1-5 mM	Reduces disulfide bonds
  PVPP	1-2%	Removes phenolic compounds

• Extraction procedure:

  • Maintain cold temperature throughout extraction (4°C)

  • Use tissue:buffer ratio of 1:3-1:5 (w/v)

  • Vortex and incubate with gentle rotation (15-30 min)

  • Centrifuge at high speed (≥12,000×g for 15 min)

  • Collect supernatant and quantify protein concentration

• Sample preparation for immunoblotting:

  • Denature proteins in Laemmli buffer at 95°C for 5 minutes

  • Load 10-50 μg total protein per lane

  • Include recombinant At4g22230 protein as positive control if available

This protocol addresses the challenges associated with plant tissue extraction, including high levels of interfering compounds and proteolytic enzymes, while preserving the native state of At4g22230 protein for optimal antibody recognition.

What troubleshooting approaches address weak or non-specific At4g22230 Antibody signals?

When encountering weak or non-specific signals with At4g22230 antibody, implement this systematic troubleshooting approach:

• For weak signals:

  • Increase antibody concentration (consider titration from 1:500 to 1:100)

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

  • Enhance signal using high-sensitivity detection substrates

  • Increase protein loading (up to 50-75 μg per lane)

  • Optimize extraction buffer to improve protein solubilization

  • Verify target protein expression under current experimental conditions

• For non-specific signals:

  • Increase blocking stringency (5% BSA or milk, consider adding 0.1% Tween-20)

  • Include additional washing steps (5× TBST washes for 10 minutes each)

  • Decrease antibody concentration

  • Pre-absorb antibody with non-specific proteins from the sample

  • Use monovalent antibody fragments to reduce background

  • Try alternative blocking agents (casein, fish gelatin)

• Validation approaches:

  • Run parallel blots with pre-immune serum as control

  • Include knockout/knockdown samples as negative controls

  • Perform peptide competition assay to confirm specificity

  • Use secondary antibody-only control to check for non-specific binding

• Optimization matrix:

  Parameter	Start with	If signal is weak	If background is high
  Antibody dilution	1:1000	Decrease to 1:500	Increase to 1:2000
  Blocking agent	5% milk	Try 5% BSA	Add 0.1% Tween-20
  Incubation time	2h at RT	Overnight at 4°C	Keep at 2h, add washes
  Detection system	Standard ECL	High-sensitivity ECL	Standard ECL

Implementing these approaches systematically will help identify the specific factors affecting At4g22230 antibody performance in your experimental system.

How can researchers design CRISPR-based approaches to validate At4g22230 Antibody specificity?

To validate At4g22230 antibody specificity using CRISPR-based approaches:

• CRISPR/Cas9 knockout design:

  • Design sgRNAs targeting early exons of AT4G22230 gene

  • Target multiple sites to ensure complete knockout

  • Consider the gene's structure to avoid off-target effects

• Controls and verification:

  • Include wild-type plants as positive controls

  • Use multiple independent knockout lines to confirm results

  • Perform RT-PCR to confirm absence of transcript

  • Sequence the targeted region to confirm gene editing

• Phenotypic characterization:

  • Compare knockout plants with wild-type for altered defense responses

  • Challenge plants with pathogens to assess immunity-related phenotypes

  • Measure expression of defense marker genes

This approach leverages methods similar to those used in functional genomics studies of Arabidopsis immunity genes, where genetic manipulation has been crucial for understanding gene function .

What comparative analysis techniques can reveal functional relationships between At4g22230 and other DEFL family proteins?

To investigate functional relationships between At4g22230 and other DEFL family proteins:

• Phylogenetic analysis:

  • Construct phylogenetic trees of DEFL family proteins to identify closely related members

  • Map conserved domains and motifs to identify functional regions

  • Compare evolutionary patterns across plant species

• Expression correlation analysis:

  • Analyze transcriptomic datasets to identify co-expressed DEFL genes

  • Create condition-specific co-expression networks

  • Use At4g22230 antibody to verify protein-level correlation with transcripts

• Functional redundancy assessment:

  • Generate single and combinatorial knockouts of At4g22230 and related DEFL genes

  • Use At4g22230 antibody to confirm protein absence in mutants

  • Assess phenotypic differences between single and multiple knockouts

• Comparative localization studies:

  • Immunolocalization with At4g22230 antibody alongside antibodies for other DEFL proteins

  • Compare subcellular and tissue localization patterns

  • Identify regions of co-localization suggesting functional relationships

• Comparative protein interaction mapping:

  Technique	Application to DEFL Family Analysis
  Yeast two-hybrid screening	Identify shared and unique interactors
  Co-immunoprecipitation	Confirm protein complexes in planta
  Protein arrays	Profile interaction with defense signaling components

This approach is informed by methods used to study protein families involved in plant immunity, where functional redundancy and specialization are common features .

How can At4g22230 Antibody facilitate research on stress-induced subcellular relocalization?

To study stress-induced subcellular relocalization of At4g22230 protein:

• Time-course immunofluorescence microscopy:

  • Treat plants with various stress conditions (pathogens, elicitors, abiotic stresses)

  • Collect samples at defined time points (0, 15, 30, 60, 180 minutes)

  • Process for immunofluorescence using the At4g22230 antibody

  • Co-label with organelle markers (nucleus, ER, Golgi, plasma membrane)

  • Capture high-resolution images and quantify protein distribution

• Subcellular fractionation with immunoblotting:

  • Isolate subcellular fractions (cytosol, membrane, nuclear, organellar)

  • Perform Western blots using At4g22230 antibody on each fraction

  • Quantify relative distribution changes following stress treatment

  • Include fraction-specific markers to confirm separation quality

• Live-cell imaging system:

  • Generate fluorescent protein fusions with At4g22230

  • Validate fusion protein functionality and localization using the At4g22230 antibody

  • Perform time-lapse imaging during stress application

  • Quantify dynamic relocalization patterns

This approach follows methodologies used to study protein relocalization during immune responses, where subcellular dynamics often play crucial roles in signal transduction .

What emerging technologies might enhance At4g22230 protein function analysis beyond current antibody-based approaches?

Several emerging technologies hold promise for advancing At4g22230 protein function analysis:

• Proximity-dependent biotinylation (BioID/TurboID):

  • Create fusion proteins to map the protein's interaction neighborhood

  • Identify transient interactions missed by traditional co-immunoprecipitation

  • Compare interactomes under different stress conditions

• CRISPR-based transcriptional modulators:

  • Use CRISPRa/CRISPRi to modulate At4g22230 expression without genetic modification

  • Create conditional expression systems to study temporal requirements

  • Apply in diverse genetic backgrounds to assess context-dependent functions

• Single-cell proteomics:

  • Analyze At4g22230 expression at single-cell resolution

  • Identify cell type-specific functions in complex tissues

  • Combine with spatial transcriptomics for integrated analysis

• Cryo-electron microscopy:

  • Determine high-resolution structures of At4g22230 alone and in complexes

  • Identify structural changes upon ligand binding or activation

  • Guide rational design of functional variants

• Synthetic biology approaches:

  • Create minimal systems reconstituting At4g22230 function

  • Design orthogonal signaling pathways incorporating At4g22230

  • Engineer enhanced or novel functions based on structural insights

These technologies complement traditional antibody-based approaches and could provide unprecedented insights into At4g22230 protein function in plant immunity and development.