At2g22941 Antibody

What is the At2g22941 gene and why is studying it important?

The At2g22941 gene encodes a protein of unknown function in Arabidopsis thaliana (mouse-ear cress), a model organism widely used in plant molecular biology. Despite limited functional annotation, genes in this chromosomal region are frequently associated with stress response pathways (e.g., drought, salinity), developmental regulation (e.g., root or leaf morphogenesis), and metabolic processes (e.g., secondary metabolite synthesis).

Studying At2g22941 is important because:

• It may provide insights into plant stress responses, which are increasingly relevant for agriculture in changing climate conditions

• Understanding its function could reveal novel developmental regulation mechanisms

• The protein may participate in metabolic pathways with potential biotechnological applications

• It represents one of many uncharacterized genes in the Arabidopsis genome, addressing a significant knowledge gap

How should I validate an At2g22941 antibody before experimental use?

Rigorous validation is essential before incorporating any antibody into experimental workflows. For At2g22941 antibody, follow these methodological steps:

• Knockout/knockdown controls: Test the antibody against wild-type and At2g22941 knockout or knockdown samples. The antibody should show significantly reduced or absent signal in the knockout/knockdown samples.

• Western blot analysis: Confirm specific detection of a protein band at the expected molecular weight. For proteins of unknown function like At2g22941, compare the observed band with the theoretical weight calculated from the amino acid sequence.

• Immunofluorescence with peptide competition: Pre-incubate the antibody with excess immunizing peptide before staining to confirm signal specificity.

• Cross-reactivity assessment: Test against closely related proteins, especially in cases where protein families share homology.

• Reproducibility across lots: When possible, validate multiple antibody lots to ensure consistent performance.

These validation steps should be documented thoroughly, as they provide the foundation for interpreting all subsequent experimental results.

What applications is the At2g22941 antibody suitable for?

Based on available information and analogous applications for plant-specific antibodies, the At2g22941 antibody can be utilized in several research applications:

The specific applications should be validated experimentally, as the At2g22941 antibody (e.g., CSB-PA651994XA01DOA) may have optimal performance in certain applications over others. Documentation for specific antibody products should be consulted for manufacturer-recommended applications and dilutions.

How can I design experiments to study protein-protein interactions involving At2g22941?

Investigating protein-protein interactions involving the poorly characterized At2g22941 protein requires a multi-faceted approach:

• Co-immunoprecipitation (Co-IP):

  • Use the At2g22941 antibody to immunoprecipitate the target protein under native conditions

  • Identify binding partners through mass spectrometry analysis

  • Validate interactions with reverse Co-IP using antibodies against putative partners

  • Include appropriate negative controls (IgG, knockout/knockdown samples)

• Proximity Labeling Approaches:

  • Generate fusion proteins with BioID or APEX2 to map the proximal interactome

  • Express these constructs in Arabidopsis protoplasts or stable transgenic lines

  • Perform streptavidin pull-down followed by mass spectrometry

• Yeast Two-Hybrid Screening:

  • Use At2g22941 as bait to screen Arabidopsis cDNA libraries

  • Validate positive interactions through orthogonal methods (Co-IP, BiFC)

• Bimolecular Fluorescence Complementation (BiFC):

  • Create fusion constructs with split fluorescent protein fragments

  • Co-express in plant cells to visualize interactions through reconstituted fluorescence

  • Include appropriate controls to rule out spontaneous complementation

The experimental design should follow principles of proper controls and statistical rigor as outlined in design of experiments methodology . Careful documentation of all experimental conditions is essential for reproducibility.

What approaches can be used to study the subcellular localization of the At2g22941 protein?

Understanding the subcellular localization of At2g22941 provides critical insights into its potential function. Several methodological approaches can be employed:

• Immunofluorescence Microscopy:

  • Fix plant tissues using 4% paraformaldehyde

  • Perform antigen retrieval if necessary

  • Incubate with At2g22941 antibody (1:100-1:500 dilution)

  • Detect with fluorescently-labeled secondary antibody

  • Co-stain with established organelle markers

  • Analyze using confocal microscopy

• Subcellular Fractionation and Western Blotting:

  • Isolate various cellular compartments (nuclei, chloroplasts, mitochondria, etc.)

  • Confirm fraction purity using established markers

  • Perform western blotting with At2g22941 antibody on each fraction

  • Quantify relative abundance across fractions

• Fluorescent Protein Fusion:

  • Generate N- and C-terminal GFP/YFP fusions with At2g22941

  • Express in Arabidopsis protoplasts or stable transgenic plants

  • Observe live cells under confocal microscopy

  • Compare both fusion orientations to rule out targeting sequence disruption

• Electron Microscopy with Immunogold Labeling:

  • Fix samples with glutaraldehyde and embed in resin

  • Prepare ultrathin sections

  • Incubate with At2g22941 antibody

  • Detect with gold-conjugated secondary antibody

  • Visualize using transmission electron microscopy

Each approach has distinct advantages and limitations. Using multiple complementary techniques provides the most robust evidence for protein localization.

How can I investigate post-translational modifications of the At2g22941 protein?

Post-translational modifications (PTMs) often regulate protein function, stability, and interactions. For a protein of unknown function like At2g22941, characterizing PTMs may provide functional insights:

• Phosphorylation Analysis:

  • Immunoprecipitate At2g22941 using validated antibody

  • Perform western blot with phospho-specific antibodies (if available)

  • Analyze by mass spectrometry to identify phosphorylation sites

  • Validate sites using phosphatase treatment controls

  • Compare phosphorylation status under different stress conditions

• Ubiquitination and SUMOylation:

  • Immunoprecipitate At2g22941 under denaturing conditions

  • Probe western blots with anti-ubiquitin or anti-SUMO antibodies

  • Use proteasome inhibitors to enhance detection of ubiquitinated forms

  • Identify modification sites using mass spectrometry

• Glycosylation Analysis:

  • Treat immunoprecipitated At2g22941 with glycosidases

  • Observe mobility shift on western blots

  • Use lectin blotting to detect specific glycan structures

  • Employ mass spectrometry for glycan profiling

• PTM-specific Enrichment Strategies:

  • Use titanium dioxide for phosphopeptide enrichment

  • Apply IMAC (Immobilized Metal Affinity Chromatography) for phosphorylated proteins

  • Employ lectin affinity chromatography for glycosylated proteins

Combining these approaches with functional studies can establish connections between specific modifications and protein function .

How should I design experiments to study At2g22941 expression under different stress conditions?

Studying At2g22941 expression under various stress conditions requires careful experimental design following these methodological principles:

• Experimental Design Framework:

  • Implement a factorial design approach to test multiple stress factors and their interactions

  • Include appropriate controls (untreated, mock treatments)

  • Ensure sufficient biological replicates (minimum n=3 for each condition)

  • Include time-course sampling when possible to capture expression dynamics

• Stress Treatment Implementation:

  • Standardize growth conditions before stress application

  • Apply controlled stress treatments (e.g., precise drought conditions through regulated watering)

  • Document all environmental parameters (light intensity, temperature, humidity)

  • Consider multiple stress intensities to establish dose-response relationships

• Expression Analysis Methods:

  • Western blotting with At2g22941 antibody for protein-level analysis

  • RT-qPCR for transcript-level analysis (design gene-specific primers)

  • Immunohistochemistry to analyze tissue-specific expression patterns

  • Consider reporter gene fusions (promoter::GUS) for spatial expression analysis

• Data Analysis Approach:

  • Apply appropriate statistical methods (ANOVA, mixed-effects models)

  • Use visualization techniques that highlight treatment effects

  • Normalize expression data to appropriate reference genes/proteins

  • Implement bioinformatic analysis to identify co-expressed genes

This methodological framework follows the principles of design of experiments (DOE) to maximize information gain while controlling for external variables .

What controls should be included when using At2g22941 antibody in immunoprecipitation experiments?

Robust immunoprecipitation (IP) experiments require comprehensive controls to ensure result validity:

For experiments identifying interaction partners, additional validation through reverse IP is recommended, where antibodies against putative partners are used to confirm co-precipitation of At2g22941 .

How can I optimize immunohistochemistry protocols for At2g22941 detection in plant tissues?

Optimizing immunohistochemistry (IHC) for the detection of At2g22941 in plant tissues requires methodical approach to multiple parameters:

• Fixation Optimization:

  • Test multiple fixatives (4% paraformaldehyde, glutaraldehyde, ethanol-based)

  • Optimize fixation time (4-24 hours) and temperature (4°C or room temperature)

  • Evaluate preservation of antigenicity vs. morphology

• Antigen Retrieval Methods:

  • Compare heat-induced epitope retrieval methods (microwave, pressure cooker)

  • Test different retrieval buffers (citrate pH 6.0, Tris-EDTA pH 9.0)

  • Optimize retrieval duration (10-30 minutes)

• Blocking Strategy:

  • Test different blocking agents (BSA, normal serum, commercial blockers)

  • Optimize blocking concentration (1-10%) and duration (1-24 hours)

  • Include detergents (0.1-0.3% Triton X-100) to enhance penetration

• Antibody Optimization:

  • Perform titration series (1:50 to 1:2000) to determine optimal concentration

  • Compare incubation conditions (4°C overnight vs. room temperature 1-4 hours)

  • Test different antibody diluents to minimize background

• Detection System Selection:

  • Compare enzymatic (HRP/DAB) vs. fluorescent detection methods

  • For fluorescence, select secondary antibody with appropriate spectral properties

  • Test signal amplification methods (tyramide signal amplification) for low-abundance targets

• Counterstaining and Mounting:

  • Select appropriate counterstains compatible with detection method

  • Use mounting media that preserves signal and prevents photobleaching

Document all optimization steps systematically to establish a reproducible protocol.

What are common reasons for weak or absent signal when using At2g22941 antibody in western blotting?

When troubleshooting weak or absent signals in western blotting with At2g22941 antibody, consider these methodological solutions:

• Protein Extraction Issues:

  • Problem: Inefficient extraction of membrane or nuclear proteins

  • Solution: Use stronger lysis buffers with appropriate detergents (RIPA, SDS-based)

  • Validation: Confirm protein extraction by Ponceau S staining or housekeeping protein detection

• Protein Abundance Limitations:

  • Problem: Low abundance of At2g22941 protein

  • Solution: Increase starting material, concentrate samples, or use immunoprecipitation

  • Validation: Load concentration series to determine detection threshold

• Epitope Accessibility Issues:

  • Problem: Epitope masking during denaturation

  • Solution: Test different denaturation conditions (varying temperatures, reducing agents)

  • Validation: Compare native vs. denaturing conditions

• Transfer Efficiency Problems:

  • Problem: Inefficient transfer of proteins to membrane

  • Solution: Optimize transfer conditions (time, voltage, buffer composition)

  • Validation: Use Ponceau S staining to confirm transfer efficiency

• Antibody Binding Limitations:

  • Problem: Suboptimal antibody concentration or incubation conditions

  • Solution: Perform antibody titration series and test different incubation times/temperatures

  • Validation: Include positive control samples with known reactivity

• Detection Sensitivity Issues:

  • Problem: Insufficient detection sensitivity

  • Solution: Use more sensitive detection methods (ECL-Plus, fluorescent secondary antibodies)

  • Validation: Compare multiple detection methods with serial dilutions

• Sample Degradation:

  • Problem: Protein degradation during preparation

  • Solution: Add protease inhibitors, maintain cold temperature, process samples quickly

  • Validation: Compare fresh vs. stored samples

Each troubleshooting step should be performed systematically, changing one variable at a time to identify the limiting factor .

How can I interpret conflicting data between protein and mRNA expression levels for At2g22941?

Discrepancies between protein and mRNA levels of At2g22941 require careful analysis and can provide valuable biological insights:

• Post-transcriptional Regulation Assessment:

  • Analyze microRNA targeting predictions for At2g22941 mRNA

  • Investigate RNA-binding protein interaction potential

  • Examine mRNA stability through actinomycin D chase experiments

  • Compare 5' and 3' UTR regulatory elements with known post-transcriptional regulators

• Translational Efficiency Analysis:

  • Perform polysome profiling to assess mRNA association with ribosomes

  • Analyze codon optimization and predict translation efficiency

  • Investigate potential upstream open reading frames (uORFs) that might regulate translation

• Protein Stability Evaluation:

  • Conduct cycloheximide chase experiments to measure protein half-life

  • Investigate ubiquitination status and proteasome-dependent degradation

  • Examine potential degrons or stability-regulating PTMs

• Technical Validation:

  • Confirm antibody specificity in knockout/knockdown systems

  • Validate RT-qPCR primer efficiency and specificity

  • Use multiple antibodies targeting different epitopes if available

  • Employ absolute quantification methods for both protein and mRNA

• Biological Context Interpretation:

  • Analyze temporal dynamics (time-course experiments)

  • Examine spatial patterns (tissue-specific differences)

  • Consider stress or developmental context-dependent regulation

Remember that protein-mRNA discrepancies are common biological phenomena reflecting the complex regulatory landscape between transcription and protein function .

How do I analyze At2g22941 expression data in the context of plant stress response pathways?

Analyzing At2g22941 expression in stress response contexts requires integrative approaches:

• Multi-omics Data Integration:

  • Correlate At2g22941 protein expression with:

    • Transcriptome data from RNA-seq experiments

    • Metabolomic profiles relevant to stress responses

    • Proteomic changes in stress-related pathways

  • Use computational approaches (WGCNA, DIABLO) to identify co-regulated networks

• Pathway Analysis:

  • Map At2g22941 to known stress response pathways using:

    • GO enrichment analysis

    • KEGG pathway mapping

    • Plant Reactome annotations

  • Use tools like MapMan to visualize expression changes in stress response context

• Comparative Expression Analysis:

  • Compare At2g22941 expression patterns with:

    • Known stress response genes

    • Transcription factors involved in stress regulation

    • Other genes in the same chromosomal region

  • Calculate correlation coefficients across multiple stress conditions

• Time-resolved Analysis:

  • Evaluate expression dynamics across stress application time course

  • Identify primary vs. secondary response patterns

  • Cluster genes with similar temporal profiles

• Mutant Phenotype Analysis:

  • Compare stress responses in wild-type vs. At2g22941 mutant plants

  • Measure physiological parameters (ROS production, stomatal conductance)

  • Evaluate stress tolerance phenotypes quantitatively

This multi-faceted approach enables robust positioning of At2g22941 within the broader context of plant stress response networks .

How can active learning approaches improve experimental design for At2g22941 functional characterization?

Active learning strategies can significantly enhance experimental efficiency in characterizing proteins like At2g22941:

• Iterative Experimental Design:

  • Begin with a small set of carefully selected conditions

  • Use initial results to guide subsequent experimental design

  • Implement Bayesian experimental design approaches to maximize information gain

  • Reduce required experiments by up to 35% compared to systematic screening approaches

• Computational Prediction Integration:

  • Employ protein structure prediction tools to guide epitope mapping

  • Use machine learning to predict protein-protein interactions

  • Implement binding site prediction algorithms

  • Validate computational predictions experimentally in targeted assays

• High-throughput Phenotyping:

  • Design plant phenotyping experiments with active learning algorithms

  • Focus on phenotypes most likely to reveal At2g22941 function

  • Adaptively select growth conditions based on initial results

  • Integrate image-based phenotyping with computational analysis

• Optimal Mutagenesis Strategy:

  • Use computational prediction to design targeted mutagenesis of At2g22941

  • Focus on conserved residues or predicted functional domains

  • Implement iterative design-build-test cycles

  • Coordinate with structural analysis to interpret results

Active learning approaches not only reduce experimental burden but can accelerate discovery by 28 steps compared to random sampling approaches , making them particularly valuable for studying proteins of unknown function like At2g22941.

What are the best approaches for studying potential post-translational modifications of At2g22941 in response to environmental stresses?

Investigating stress-responsive PTMs of At2g22941 requires specialized methodologies:

• Quantitative PTM Profiling:

  • Implement SILAC or TMT labeling for comparative PTM analysis

  • Apply targeted mass spectrometry (SRM/MRM) for specific modification sites

  • Use parallel reaction monitoring for higher sensitivity detection

  • Compare PTM profiles across multiple stress conditions

• Site-specific Mutagenesis:

  • Generate phosphomimetic mutants (S/T to D/E) and phospho-null mutants (S/T to A)

  • Create lysine mutants (K to R) to block ubiquitination/SUMOylation

  • Introduce these mutations in native gene context using CRISPR/Cas9

  • Evaluate phenotypic consequences under stress conditions

• Kinase/Enzyme Identification:

  • Perform in vitro kinase assays with recombinant At2g22941

  • Use kinase inhibitor panels to identify responsible kinase families

  • Apply proximity labeling approaches to identify modifying enzymes

  • Conduct yeast two-hybrid screens with conditionally expressed constructs

• Dynamic PTM Visualization:

  • Develop phosphorylation-specific antibodies for At2g22941

  • Apply SDS-PAGE with Phos-tag™ acrylamide for mobility shift detection

  • Implement FRET-based biosensors for real-time PTM monitoring

  • Use split-GFP systems coupled to modification-dependent interactions

These approaches provide complementary information on the PTM landscape of At2g22941 and its regulation under stress conditions, similar to strategies used for other regulatory proteins .

How can I design a comprehensive study to elucidate the role of At2g22941 in plant development and stress response?

A comprehensive functional characterization of At2g22941 requires a multi-disciplinary approach:

• Genetic Resources Development:

  • Generate multiple allelic variants (knockout, knockdown, overexpression)

  • Create tissue-specific and inducible expression systems

  • Develop fluorescent protein fusions for live imaging

  • Establish CRISPR/Cas9 genome editing for precise mutations

• Multi-scale Phenotyping:

  • Analyze developmental phenotypes across complete life cycle

  • Implement high-throughput image-based phenotyping

  • Conduct detailed cellular and subcellular analysis

  • Perform stress tolerance assays under multiple conditions

• Multi-omics Integration:

  • Compare transcriptomes (RNA-seq) between wild-type and mutant lines

  • Analyze proteomes and phosphoproteomes under normal and stress conditions

  • Conduct metabolomic profiling to identify affected pathways

  • Perform chromatin immunoprecipitation if nuclear localization is observed

• Interactome Mapping:

  • Conduct yeast two-hybrid and split-ubiquitin screens

  • Implement proximity labeling approaches (BioID, APEX)

  • Perform co-immunoprecipitation with At2g22941 antibody

  • Validate key interactions through multiple orthogonal methods

• Evolutionary Analysis:

  • Compare At2g22941 with orthologs across plant species

  • Analyze selection pressure on different protein domains

  • Investigate gene duplication and diversification patterns

  • Correlate evolutionary conservation with functional elements

This comprehensive approach follows design of experiments principles and integrates techniques from molecular biology, genetics, biochemistry, and computational biology to develop a complete understanding of At2g22941 function.