ATS3B Antibody

What is ATS3B and why is it significant for plant biology research?

ATS3B (Embryo-specific protein 3B) is a protein encoded by the AT5G62200 gene in Arabidopsis thaliana. This protein was initially identified through differential display of mRNA as an embryo-specific gene . The significance of ATS3B stems from its roles in:

• Embryonic development in Arabidopsis

• Stress response mechanisms

• Stomatal regulation (particularly closure)

• Potential roles in plant defense against pathogens

Research has demonstrated that ATS3B interacts with other proteins such as ArathEULS3 (a lectin involved in drought stress response) in closed stomata, suggesting a role in stress-related signaling pathways . Understanding ATS3B function provides insights into fundamental plant developmental and stress adaptation mechanisms.

What detection methods can be used with ATS3B antibodies?

Based on available research antibodies and protocols, ATS3B antibodies can be utilized in several detection methods:

• Western Blotting (WB): For detecting the ATS3B protein in tissue lysates and determining protein size/expression levels

• Immunohistochemistry (IHC): For localizing ATS3B in tissue sections

• Enzyme-Linked Immunosorbent Assay (ELISA): For quantitative measurement of ATS3B

• Immunocytochemistry (ICC): For subcellular localization studies

The selection of method depends on your specific research question. For instance, when investigating protein-protein interactions involving ATS3B, bimolecular fluorescence complementation experiments have been successfully employed to confirm interactions between ArathEULS3 and ATS3B in closed stomata of Nicotiana benthamiana plants .

How should researchers select and validate an ATS3B antibody for their experiments?

Proper antibody selection and validation are critical for reliable results. Follow these methodological steps:

• Antibody Type Selection: Consider polyclonal antibodies for higher sensitivity (like those raised in rabbits against ATS3B ). Monoclonal antibodies provide higher specificity but may be less available for ATS3B.

• Species Reactivity: Verify that the antibody specifically recognizes ATS3B from your species of interest. Most available antibodies are designed for Arabidopsis thaliana .

• Application Validation: Confirm the antibody has been validated for your intended application (WB, IHC, ICC). Look for published studies and validation data from manufacturers.

• Epitope Information: Review the immunogen information. For ATS3B antibodies, some are generated against recombinant Arabidopsis thaliana ATS3B protein .

• Controls for Validation:

  • Positive control: Use tissues known to express ATS3B (such as Arabidopsis embryonic tissues)

  • Negative control: Use tissues where ATS3B is not expressed or ATS3B knockout lines

  • Blocking peptide: To confirm specificity of signal

  • Cross-reactivity testing: Especially important when working with related proteins

• Documentation: Always record antibody catalog numbers, lot numbers, and dilutions used for reproducibility.

What are the optimal protocols for using ATS3B antibodies in co-immunoprecipitation studies?

When designing co-immunoprecipitation (Co-IP) experiments to study ATS3B interactions:

• Sample Preparation:

  • Fresh plant tissue is preferable (embryonic tissue or tissues where ATS3B is known to be expressed)

  • Use a gentle lysis buffer (e.g., 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.5% NP-40, protease inhibitors)

  • Maintain cold conditions throughout to preserve protein complexes

• Antibody Binding:

  • Pre-clear lysate with protein A/G beads to reduce nonspecific binding

  • Incubate cleared lysate with ATS3B antibody (typically 2-5 μg of antibody per 500 μg of protein)

  • Allow binding to occur overnight at 4°C with gentle rotation

• Controls:

  • Include a negative control with IgG from the same species as the ATS3B antibody

  • Include a sample from ATS3B knockout or knockdown plants

  • Consider a reciprocal Co-IP when possible (using antibodies against suspected interaction partners)

• Verification of Interactions:

  • Confirm interactions with alternative methods such as bimolecular fluorescence complementation, as demonstrated for ATS3B and ArathEULS3

  • For suspected novel interactions, tandem affinity purification followed by mass spectrometry can be employed

• Analysis of Results:

  • Analyze by Western blotting, probing for both ATS3B and the interacting protein

  • Consider protein complex stability when interpreting negative results

How can researchers effectively use ATS3B antibodies to study its role in stomatal regulation?

Based on published research showing ATS3B's involvement in stomatal closure , the following methodological approach is recommended:

• Tissue Selection:

  • Use leaf epidermal peels or intact leaves from plants at appropriate developmental stages

  • Consider comparing wild-type, ATS3B overexpression, and ATS3B knockdown/knockout lines

• Immunolocalization Protocol:

  • Fix tissue samples with 4% paraformaldehyde

  • Permeabilize with 0.1% Triton X-100

  • Block with 3% BSA

  • Incubate with ATS3B primary antibody (optimal dilution typically 1:100 to 1:500)

  • Use appropriate fluorophore-conjugated secondary antibody

  • Include DAPI staining for nuclei visualization

• Stimulus Testing:

  • Apply relevant stimuli (ABA, drought, pathogen-associated molecular patterns)

  • Perform time-course experiments to capture dynamic changes

• Quantification Methods:

  • Measure stomatal aperture changes

  • Quantify ATS3B protein levels and localization before and after stimulus

  • Correlate with functional stomatal responses

• Combined Approaches:

  • Use bimolecular fluorescence complementation to visualize protein interactions in guard cells

  • Consider co-staining with markers for relevant cellular compartments

What are the most common issues when using ATS3B antibodies and how can they be resolved?

How can researchers confirm the specificity of ATS3B antibody signal in their experiments?

To methodically confirm antibody specificity:

• Genetic Approaches:

  • Use tissues from ATS3B knockout mutants (such as SALK_133822C line ) as a negative control

  • Compare wild-type with ATS3B-overexpressing plants to observe proportional signal increases

• Biochemical Approaches:

  • Perform peptide competition assays using the immunogenic peptide

  • Conduct immunoprecipitation followed by mass spectrometry to confirm the identity of the pulled-down protein

• Multiple Antibody Validation:

  • Use antibodies raised against different epitopes of ATS3B

  • Compare results from monoclonal and polyclonal antibodies when available

• Cross-species Validation:

  • Test the antibody in closely related species where the epitope is conserved

  • Compare expression patterns with known tissue-specific expression data

• Correlation with mRNA Expression:

  • Compare protein detection patterns with mRNA expression (RT-PCR or RNA-seq data)

  • Verify temporal and spatial expression patterns match known patterns for ATS3B

How can ATS3B antibodies be employed to investigate the protein's role in stress signaling pathways?

To investigate ATS3B's role in stress signaling:

• Stress Treatment Experimental Design:

  • Expose plants to relevant stresses (drought, pathogens, ABA)

  • Collect samples at multiple time points (0, 1, 3, 6, 12, 24 hours)

  • Process parallel samples for protein extraction and microscopy

• Protein Complex Dynamics Analysis:

  • Conduct co-immunoprecipitation with ATS3B antibodies before and after stress

  • Analyze interacting partners by mass spectrometry

  • Compare interaction profiles under different stress conditions

• Phosphorylation Status Analysis:

  • Perform immunoprecipitation with ATS3B antibodies

  • Analyze phosphorylation status using phospho-specific antibodies or mass spectrometry

  • Correlate changes with stress response timing

• Subcellular Localization Changes:

  • Use immunofluorescence microscopy to track ATS3B localization during stress

  • Co-stain with organelle markers to determine translocation events

  • Quantify changes in localization patterns

• Correlation with Physiological Responses:

  • Measure stomatal aperture, transpiration rates, or pathogen resistance

  • Correlate with ATS3B protein levels, modifications, and interactions

  • Compare wild-type responses with those in ATS3B-modified plants

Research has shown that during bacterial infection of Arabidopsis thaliana plants, there was a 6-fold increase in transcript levels for ArathEULS3, which interacts with ATS3B . This suggests ATS3B may play a role in pathogen response pathways.

What methodological approaches can be used to study the interaction between ATS3B and ArathEULS3 in stomatal regulation?

Based on published research on ATS3B-ArathEULS3 interaction , the following methodological approaches are recommended:

• In vivo Interaction Visualization:

  • Bimolecular fluorescence complementation (BiFC): Split YFP or similar fluorescent protein between ATS3B and ArathEULS3, observe reconstituted fluorescence in guard cells

  • FRET analysis: Tag proteins with compatible fluorophores to measure energy transfer indicating close proximity

  • Split-luciferase complementation assays in transiently transformed leaves

• Dynamic Interaction Analysis:

  • Apply ABA or pathogen-associated molecular patterns and observe changes in interaction

  • Perform time-course studies following stimulus application

  • Correlate interaction intensity with stomatal aperture changes

• Domain Mapping:

  • Generate truncated versions of both proteins to identify interaction domains

  • Perform site-directed mutagenesis of key residues

  • Verify effects on interaction and stomatal function

• Functional Analysis:

  • Compare stomatal responses in wild-type, single mutants (ATS3B or ArathEULS3), and double mutants

  • Perform complementation studies with mutated versions of either protein

  • Correlate interaction strength with functional outputs

• Protein Modification Effects:

  • Investigate post-translational modifications affecting the interaction

  • Test conditions that might regulate the interaction (pH, calcium levels, redox state)

Studies have confirmed interactions between ArathEULS3 and ATS3B in closed stomata of Nicotiana benthamiana plants using BiFC experiments . Plants with reduced ArathEULS3 expression exhibited aberrant ABA-induced stomatal closure compared to overexpressing and control plants, suggesting a functional relationship between these interacting proteins.

How should researchers interpret varying signal intensities of ATS3B across different plant tissues and developmental stages?

When analyzing variations in ATS3B signal across tissues and developmental stages:

• Expression Pattern Analysis:

  • Compare observed protein levels with known mRNA expression patterns

  • ATS3B was initially identified as embryo-specific , so higher expression is expected in embryonic tissues

  • Unexpected expression in non-embryonic tissues may indicate novel functions or stress responses

• Developmental Timeline Considerations:

  • Document precise developmental stages using standardized growth stage definitions

  • Consider normal developmental regulation when interpreting apparent changes

  • Create developmental expression maps to identify critical transition points

• Standardization Approaches:

  • Always normalize to appropriate loading controls (housekeeping proteins)

  • Consider using recombinant ATS3B standards for absolute quantification

  • Include reference tissues in each experiment for relative comparisons

• Tissue-Specific Variables:

  • Account for tissue-specific extraction efficiency differences

  • Consider cell-type heterogeneity within tissue samples

  • Use cell-type specific markers when performing immunolocalization

• Biological vs. Technical Variation:

  • Distinguish between biological replicates (different plants) and technical replicates

  • Use statistical methods appropriate for the experimental design

  • Report both types of variation when presenting results

Research has shown that ATS3B exhibits spatial expression patterns similar to Arabidopsis seed storage protein genes , which should be considered when interpreting expression data.

How can researchers determine if detected post-translational modifications of ATS3B are biologically significant?

To evaluate the biological significance of ATS3B post-translational modifications (PTMs):

• Conservation Analysis:

  • Examine if the modified residues are conserved across species

  • Compare with related proteins (such as ATS3A) to identify conserved modification sites

  • Higher conservation suggests functional importance

• Correlation with Biological Events:

  • Track modification status during development and stress responses

  • Correlate changes with phenotypic outputs (stomatal closure, stress resistance)

  • Establish temporal relationships between modification and downstream events

• Functional Impact Assessment:

  • Generate site-directed mutants that mimic or prevent modification

  • Test mutants for altered function in relevant assays

  • Assess impacts on protein-protein interactions, particularly with known partners like ArathEULS3

• Enzymatic Regulation:

  • Identify enzymes responsible for the modifications

  • Use inhibitors or genetic approaches to modulate these enzymes

  • Observe resulting changes in ATS3B function

• Structural Considerations:

  • Model the impact of modifications on protein structure

  • Predict effects on interaction surfaces or functional domains

  • Test predictions through experimental approaches

While specific information on ATS3B post-translational modifications is limited in the provided search results, research in plant biology often reveals that PTMs play crucial roles in regulating protein function, especially in stress responses and developmental transitions.