At4g18593 Antibody

What is AT4G18593 and why is it significant in Arabidopsis research?

AT4G18593 encodes a dual specificity protein phosphatase-like protein in Arabidopsis thaliana (mouse-ear cress) . This protein belongs to a family of phosphatases that can dephosphorylate both serine/threonine and tyrosine residues, potentially playing important roles in cellular signaling pathways including stress responses and development. The significance lies in understanding phosphorylation-dependent signaling networks in plants, which remain less characterized than their animal counterparts.

What methods are most effective for validating AT4G18593 antibody specificity?

Effective validation requires multiple complementary approaches:

• Western blot analysis comparing wild-type and knockout/knockdown plants

• Immunoprecipitation followed by mass spectrometry to confirm target identity

• Preabsorption tests with recombinant AT4G18593 protein to confirm epitope specificity

• Testing in multiple Arabidopsis tissues to verify expression patterns match transcriptome data

• Including appropriate positive controls (such as tagged recombinant AT4G18593) and negative controls (such as tissues from knockout lines)

Note that cross-reactivity testing against related phosphatases is essential due to sequence conservation among dual-specificity phosphatase family members.

How should I design immunolocalization experiments to detect AT4G18593 in plant tissues?

For successful immunolocalization of AT4G18593:

• Fixation protocol: Use 4% paraformaldehyde in PBS for tissue preservation while maintaining antigenicity

• Tissue preparation: Consider both cross-sections and longitudinal sections of different organs

• Antigen retrieval: Mild citrate buffer (pH 6.0) heat treatment may improve epitope accessibility

• Blocking: 5% BSA with 0.3% Triton X-100 in PBS minimizes non-specific binding

• Primary antibody: Optimal dilution must be empirically determined (typically 1:100-1:500)

• Controls: Include:

  • Primary antibody omission control

  • Preimmune serum control

  • AT4G18593 mutant/knockout tissue control

• Detection: Secondary antibodies conjugated to fluorophores compatible with plant autofluorescence spectra

• Counterstain: DAPI for nuclear visualization

• Confocal microscopy: Z-stack acquisition to visualize subcellular localization

The key for successful visualization is thorough optimization of permeabilization conditions, which may vary between different plant tissues.

What are the optimal protocols for using AT4G18593 antibodies in chromatin immunoprecipitation (ChIP) assays?

For optimal ChIP assays with AT4G18593 antibodies:

• Crosslinking: 1% formaldehyde for 10 minutes at room temperature under vacuum

• Tissue disruption: Grinding in liquid nitrogen followed by nuclear isolation

• Chromatin shearing: Optimize sonication conditions (typically 15-30 second pulses for 10-15 cycles) to achieve fragments of 200-500 bp

• Pre-clearing: Incubate chromatin with protein A/G beads and non-immune IgG

• Immunoprecipitation: Incubate with anti-AT4G18593 antibody overnight at 4°C

• Washing: Use increasingly stringent buffers (Low salt → High salt → LiCl → TE)

• Elution and reverse crosslinking: 65°C for 6 hours

• DNA purification: Phenol-chloroform extraction or commercial kits

• Validation: qPCR of purified DNA against known phosphatase-regulated genes

The critical parameter is antibody quality - ensure the antibody recognizes native (non-denatured) AT4G18593 protein .

Why might I observe inconsistent results when using AT4G18593 antibodies in Western blots?

Inconsistent Western blot results may stem from several factors:

• Protein extraction method:

  • Phosphatases are sensitive to extraction conditions

  • Use phosphatase inhibitor cocktails (e.g., sodium fluoride, sodium orthovanadate)

  • Extract in denaturing conditions (with SDS) to prevent proteolysis

• Sample processing:

  • Avoid repeated freeze-thaw cycles

  • Maintain cold chain throughout

  • Process samples rapidly to minimize degradation

• Transfer issues:

  • Optimize transfer time based on protein size (~36 kDa)

  • Consider semi-dry vs. wet transfer optimization

• Detection sensitivity:

  • Enhanced chemiluminescence may be required for low abundance proteins

  • Consider fluorescent secondary antibodies for quantitative analysis

• Post-translational modifications:

  • Phosphorylation status of AT4G18593 itself may affect antibody recognition

  • Consider phosphatase treatment of extracts as control

The most common cause of inconsistency is variation in extraction protocols - standardize your lysis buffer and extraction procedure for all comparative studies.

How can I minimize background when using AT4G18593 antibodies in immunofluorescence microscopy of Arabidopsis tissues?

To minimize background in immunofluorescence:

• Optimize fixation:

  • Over-fixation can create artifactual cross-reactions

  • Under-fixation can compromise tissue morphology

  • Test multiple fixation times (15 min to 2 hours)

• Enhanced blocking:

  • Extend blocking time to 2+ hours at room temperature

  • Include 0.1% Tween-20 in blocking buffer

  • Consider adding 5% normal serum from the species of secondary antibody

  • For recalcitrant tissues, add 1% glycine to quench aldehyde groups from fixative

• Antibody dilution:

  • Titrate primary antibody (1:50 to 1:2000)

  • Use antibody dilution buffer containing 0.05% Tween-20

• Washing protocol:

  • Increase number of washes (5-6 times)

  • Extend wash duration (15-20 minutes each)

  • Use gentle agitation during washes

• Autofluorescence reduction:

  • Pre-treat sections with 0.1% sodium borohydride

  • Include 0.01% Pontamine Fast Scarlet 4B for root tissues

  • Image using spectral unmixing when available

The tissue-specific optimization of permeabilization is often overlooked but crucial - root tissues require different permeabilization than leaf tissues.

How can AT4G18593 antibodies be employed in studying protein-protein interactions in stress response pathways?

For studying protein-protein interactions:

• Co-immunoprecipitation approaches:

  • Use crosslinking reagents (DSP, formaldehyde) to capture transient interactions

  • Apply tandem affinity purification with AT4G18593 antibodies

  • Compare interaction profiles under normal vs. stress conditions

  • Verify interactions with reciprocal co-IP experiments

• Proximity-based labeling:

  • Express BioID or TurboID fusions with AT4G18593

  • Validate with antibody-based detection of biotinylated proteins

  • Compare interactome under different stress conditions

• Fluorescence-based interaction assays:

  • FRET between fluorescently-tagged AT4G18593 and potential partners

  • Use antibodies to verify native protein interactions by PLA (Proximity Ligation Assay)

• Dynamic interaction studies:

  • Time-course experiments following stress application

  • Subcellular fractionation to determine compartment-specific interactions

  • Phosphorylation-dependent interactions before/after phosphatase inhibitor treatment

Recent research suggests AT4G18593 may participate in stress-responsive phosphorylation cascades, making interaction studies particularly valuable for understanding signaling network topology.

What strategies can be employed to analyze post-translational modifications of AT4G18593 using specific antibodies?

For PTM analysis:

• Phosphorylation-specific antibody approaches:

  • Generate phospho-specific antibodies against predicted sites

  • Use phosphatase inhibitors during extraction

  • Compare signals with and without phosphatase treatment

  • Validate with mass spectrometry-identified phosphorylation sites

• Combined approaches:

  • Immunoprecipitate with AT4G18593 antibody followed by:

    • Western blotting with anti-phosphoserine/threonine antibodies

    • Ubiquitination detection with anti-ubiquitin antibodies

    • SUMOylation assessment with anti-SUMO antibodies

• Stimulus-dependent modification:

  • Track PTM changes following hormone treatment

  • Compare PTM patterns during development

  • Analyze stress-induced modifications

• Functional correlation:

  • Correlate PTM status with enzymatic activity using in-gel phosphatase assays

  • Compare subcellular localization with PTM status

  • Assess protein stability in relation to modification state

The challenge with plant PTM analysis is typically low abundance of the modified form - consider enrichment strategies such as phosphopeptide enrichment prior to analysis.

How can AT4G18593 antibodies be integrated with proteomics approaches to study plant stress responses?

Integration with proteomics involves:

• Immunoaffinity enrichment:

  • Use AT4G18593 antibodies conjugated to beads for pulldown

  • Apply to total plant extracts from control and stress conditions

  • Identify co-purifying proteins by mass spectrometry

  • Quantify changes in interaction partners between conditions

• Targeted proteomics:

  • Develop multiple reaction monitoring (MRM) assays

  • Validate findings with antibody-based detection

  • Quantify protein level changes in different tissues/conditions

• Spatial proteomics:

  • Combine tissue-specific extraction with antibody-based verification

  • Use laser capture microdissection followed by immunoblotting

  • Correlate with fluorescence microscopy data

• Temporal dynamics:

  • Time-course experiments with immunoprecipitation at multiple timepoints

  • Pulse-chase experiments coupled with antibody-based detection

  • Integrate with transcriptomics for multi-omics analysis

Recent studies suggest differential phosphorylation networks activated under abiotic stress conditions, making AT4G18593 a valuable target for understanding stress adaptation mechanisms.

What considerations are important when developing CRISPR-edited plant lines for validating AT4G18593 antibody specificity?

For CRISPR validation lines:

• Gene editing strategy:

  • Design guide RNAs targeting early exons

  • Create epitope-disrupting mutations rather than complete knockouts

  • Generate multiple independent lines with different indel patterns

  • Create C-terminal tag knock-in lines as positive controls

• Validation workflow:

  • Confirm edits by sequencing

  • Assess transcript levels by RT-qPCR

  • Compare protein detection across multiple tissues

  • Perform side-by-side Western blots of wild-type and edited lines

• Controls and considerations:

  • Include wild-type biological replicates

  • Use tissue-matched samples

  • Consider developmental stage effects on expression

  • Assess potential compensatory upregulation of related genes

• Advanced validation:

  • Perform epitope mapping to determine exact antibody binding site

  • Create synthetic peptide competition assays

  • Compare multiple commercial antibodies if available

The most rigorous validation approach combines complete knockouts with specific epitope mutations and tagged knock-in lines in the same genetic background.

How can newly developed proximity labeling approaches be combined with AT4G18593 antibodies for in vivo interaction studies?

Combining proximity labeling with AT4G18593 antibodies:

• TurboID or BioID fusion constructs:

  • Create N- and C-terminal TurboID fusions with AT4G18593

  • Express under native or inducible promoters

  • Verify fusion protein expression and localization using AT4G18593 antibodies

  • Optimize biotin labeling conditions for plant tissues

• Validation strategy:

  • Use AT4G18593 antibodies to confirm fusion protein expression

  • Perform streptavidin pulldowns of biotinylated proteins

  • Verify known interactors by immunoblotting with specific antibodies

  • Identify novel interactors by mass spectrometry

• Application scenarios:

  • Compare interactomes across different stress conditions

  • Study developmental changes in interaction networks

  • Identify tissue-specific interaction partners

  • Track dynamic responses to hormonal treatments

• Methodological refinements:

  • Use split-TurboID for binary interaction verification

  • Combine with conditional systems for temporal control

  • Integrate with metabolic labeling for protein turnover assessment

This approach has recently revealed unexpected connections between phosphatase networks and hormone signaling pathways in Arabidopsis.

What are the considerations for using AT4G18593 antibodies in single-cell protein analysis of plant tissues?

For single-cell protein analysis:

• Sample preparation:

  • Optimize protoplast isolation protocols for target tissues

  • Minimize stress responses during preparation

  • Consider fixation methods compatible with antibody recognition

  • Maintain cell viability for functional assays

• Detection methods:

  • Flow cytometry with fluorescently-labeled secondary antibodies

  • Imaging flow cytometry for subcellular localization

  • In situ proximity ligation assay for protein-protein interactions

  • Single-cell Western blotting for size verification

• Validation requirements:

  • Confirm specificity in whole-tissue extracts

  • Use knockout/knockdown protoplasts as negative controls

  • Assess potential protoplasting artifacts

  • Compare to in situ detection in intact tissues

• Data analysis considerations:

  • Account for cell-type heterogeneity

  • Normalize to appropriate reference proteins

  • Correlate with single-cell transcriptomics data

  • Apply computational approaches for trajectory inference

The key challenge is balancing gentle cell isolation with effective permeabilization for antibody access while maintaining native protein interactions.

How transferable are AT4G18593 antibodies for detecting homologous proteins in other plant species?

Transferability considerations:

• Sequence conservation analysis:

  • Perform sequence alignments of AT4G18593 with homologs

  • Identify epitope conservation across species

  • Consider raising antibodies against highly conserved regions

• Validation requirements:

  • Test antibodies on recombinant homologous proteins

  • Perform Western blots on multiple species

  • Include competition assays with Arabidopsis recombinant protein

  • Verify signal absence in knockout/knockdown lines when available

• Species-specific optimizations:

  • Adjust extraction buffers for different tissue compositions

  • Modify fixation protocols for immunohistochemistry

  • Optimize blocking agents to reduce non-specific binding

  • Consider tissue-specific extraction modifications

• Experimental considerations:

  • Load equal total protein amounts (rather than equal tissue mass)

  • Include positive controls from Arabidopsis

  • Consider differences in protein abundance between species

  • Account for potential post-translational modification differences

Antibodies raised against conserved catalytic domains typically show better cross-species reactivity than those targeting variable regions or plant-specific domains.

What methodological adaptations are needed when applying AT4G18593 antibodies in non-model plant systems?

For non-model plant applications:

• Extraction protocol modifications:

  • Adjust buffer composition for species-specific compounds

  • Account for different secondary metabolite profiles

  • Increase protease/phosphatase inhibitor concentrations

  • Consider specific removal of interfering compounds

• Detection adaptations:

  • Optimize protein loading amounts (typically higher for cross-species detection)

  • Extend antibody incubation times

  • Consider more sensitive detection methods (chemiluminescence vs. colorimetric)

  • Test multiple antibody dilutions

• Validation approaches:

  • Peptide competition assays to demonstrate specificity

  • Immunoprecipitation followed by mass spectrometry

  • Expression of target protein in heterologous systems

  • Correlation with transcript levels when feasible

• Immunolocalization considerations:

  • Optimize fixation protocols for different tissue compositions

  • Adjust permeabilization for species-specific cell wall differences

  • Increase blocking stringency to reduce background

  • Include comprehensive controls for autofluorescence

When working with woody species, consider the presence of phenolic compounds that may interfere with antibody binding and require additional extraction steps with PVPP or β-mercaptoethanol.