At3g59230 Antibody

What is the At3g59230 gene and why is it significant in Arabidopsis research?

At3g59230 encodes a putative F-box/LRR-repeat protein in Arabidopsis thaliana. F-box proteins generally function as part of SCF (Skp, Cullin, F-box) complexes that mediate protein-protein interactions and are critical in ubiquitin-mediated protein degradation pathways. The LRR (Leucine-Rich Repeat) domain typically facilitates specific protein-protein interactions. Understanding At3g59230's function can provide insights into how plants regulate protein degradation and cellular signaling in response to various stimuli, including environmental stresses. While not extensively characterized in the provided literature, similar F-box proteins have been implicated in hormone signaling, development, and stress responses in plants .

What are the optimal storage conditions for At3g59230 antibodies?

At3g59230 antibodies should be stored according to the manufacturer's specifications. Typically, they are preserved in a buffer containing 0.03% Proclin 300 (as a preservative) and 50% Glycerol in 0.01M Phosphate Buffered Saline (PBS) at pH 7.4. These antibodies are generally shipped with ice packs and should be stored at -20°C for long-term preservation. Avoid repeated freeze-thaw cycles, which can degrade antibody quality. For working solutions, store at 4°C for up to one month. Always centrifuge briefly before opening the vial to ensure the antibody solution is at the bottom of the tube.

What experimental techniques can At3g59230 antibodies be used for?

At3g59230 antibodies can be utilized in various experimental techniques including:

• Western blotting (immunoblotting): For detecting and quantifying At3g59230 protein expression levels

• Immunoprecipitation (IP): To isolate At3g59230 protein complexes

• Chromatin immunoprecipitation (ChIP): If investigating potential DNA-protein interactions

• Immunohistochemistry (IHC): To visualize protein localization in plant tissues

• Enzyme-linked immunosorbent assay (ELISA): For quantitative protein detection

When designing experiments, researchers should validate the antibody for their specific application and plant tissue, as antibody performance can vary between techniques and experimental conditions .

How can I validate the specificity of an At3g59230 antibody?

Validating antibody specificity is critical for reliable experimental results. For At3g59230 antibody validation, consider these approaches:

• Positive and negative controls: Compare wild-type Arabidopsis tissues with At3g59230 knockout/knockdown lines.

• Blocking peptide experiments: Pre-incubate the antibody with the immunizing peptide before application to confirm signal specificity.

• Western blot analysis: Verify that the detected band matches the predicted molecular weight of At3g59230 protein.

• Multiple antibody approach: Use antibodies generated against different epitopes of At3g59230.

• Cross-reactivity testing: Test the antibody against related F-box proteins to ensure specificity.

Follow established protocols for antibody validation similar to those used for other plant proteins, adjusting buffer conditions and experimental parameters as needed for plant-specific considerations .

How does At3g59230 expression change in response to geomagnetic field variations?

Transcriptomic analyses have revealed that exposure to Near Null Magnetic Field (NNMF) conditions induces differential gene expression patterns in Arabidopsis thaliana. While At3g59230 was not specifically highlighted in the provided gene expression tables, other F-box/LRR-repeat family proteins have shown significant modulation under magnetic field variations. For instance, similar F-box proteins showed tissue-specific expression changes, with some demonstrating upregulation in roots and downregulation in shoots under NNMF conditions .

Research methodology would involve:

• Growing Arabidopsis seedlings under controlled conditions

• Exposing plants to normal Geomagnetic Field (GMF) and NNMF conditions

• Extracting RNA from roots and shoots separately

• Performing quantitative RT-PCR or microarray analysis to measure At3g59230 expression

• Validating expression changes at the protein level using At3g59230 antibodies

When investigating At3g59230 responses to geomagnetic field variations, researchers should design time-course experiments (10 min to 96 h exposure) to capture both early and late responses .

What is the relationship between At3g59230 and nitrogen starvation response in Arabidopsis?

While direct evidence linking At3g59230 to nitrogen starvation responses was not explicitly provided in the search results, F-box proteins often play roles in nutrient sensing and adaptation mechanisms. To investigate this relationship, researchers could:

• Compare At3g59230 expression levels between nitrogen-sufficient and nitrogen-starved plants using quantitative RT-PCR

• Use stem-loop RT-PCR to identify any miRNAs that might regulate At3g59230 expression under nitrogen starvation

• Generate transgenic Arabidopsis plants overexpressing or silencing At3g59230 to assess phenotypic responses to nitrogen limitation

• Employ At3g59230 antibodies to quantify protein levels and post-translational modifications during nitrogen starvation

• Identify potential interaction partners that change under nitrogen starvation using co-immunoprecipitation with At3g59230 antibodies

The methodology would follow similar approaches to those used in miRNA identification and target prediction studies, using tools like WMD3 software package for predicting regulatory relationships .

What techniques can be used to identify proteins that interact with At3g59230?

As an F-box/LRR-repeat protein, At3g59230 likely functions through protein-protein interactions. To identify interaction partners:

• Yeast Two-Hybrid (Y2H) Screening: Express At3g59230 as bait to screen an Arabidopsis cDNA library.

• Co-Immunoprecipitation (Co-IP): Use At3g59230 antibodies to pull down protein complexes from plant extracts, followed by mass spectrometry identification.

• Bimolecular Fluorescence Complementation (BiFC): Test direct interactions with candidate proteins in planta.

• Proximity-Dependent Biotin Identification (BioID): Fuse a biotin ligase to At3g59230 to biotinylate nearby proteins for subsequent purification and identification.

• Tandem Affinity Purification (TAP): Express tagged At3g59230 in Arabidopsis and purify intact protein complexes.

For Co-IP experiments specifically, optimize extraction buffers to preserve interactions while minimizing non-specific binding. Consider using mild detergents (0.1-0.5% NP-40 or Triton X-100) and including protease inhibitors. Cross-linking with formaldehyde prior to extraction can help stabilize transient interactions .

What are the key considerations for designing immunohistochemistry experiments with At3g59230 antibodies?

When designing immunohistochemistry experiments to localize At3g59230 protein in plant tissues:

• Tissue Fixation: Use 4% paraformaldehyde or a plant-specific fixative optimized for preserving protein epitopes while maintaining tissue structure.

• Antigen Retrieval: Test different antigen retrieval methods (heat-induced, enzymatic, or pH-based) to optimize epitope accessibility.

• Blocking Parameters: Use 3-5% BSA or normal serum from the species of the secondary antibody to minimize background.

• Antibody Dilution: Determine optimal primary (At3g59230) and secondary antibody dilutions through titration experiments (typically starting at 1:100 to 1:1000).

• Controls: Include:

  • Negative controls (no primary antibody)

  • Positive controls (tissues known to express At3g59230)

  • Peptide competition controls

  • Parallel staining with At3g59230 knockout/knockdown tissues

Consider tissue-specific autofluorescence when selecting detection methods, and optimize incubation times and temperatures for each step of the protocol .

How can I troubleshoot weak or absent signals when using At3g59230 antibodies in Western blots?

When encountering weak or absent signals in Western blots using At3g59230 antibodies, consider the following troubleshooting approaches:

Additionally, ensure your extraction buffer preserves the At3g59230 protein (consider adding protease inhibitors) and optimize SDS-PAGE conditions for the expected molecular weight of At3g59230 .

What are the recommended protocols for quantifying At3g59230 expression changes in response to environmental stresses?

To quantify At3g59230 expression changes in response to environmental stresses, a comprehensive approach combining both transcript and protein analysis is recommended:

For transcript-level analysis:

• Extract total RNA using Trizol reagent from stress-treated and control plants

• Perform DNase I digestion to remove genomic DNA contamination

• Synthesize cDNA using oligo(dT)18 primers

• Conduct quantitative RT-PCR using At3g59230-specific primers

• Normalize expression data to a stable reference gene such as ACT2 (At3g18780)

For protein-level analysis:

• Extract total protein using a buffer optimized for plant tissues (e.g., containing 50mM Tris-HCl pH 7.5, 150mM NaCl, 1% Triton X-100, protease inhibitors)

• Quantify protein concentration using Bradford or BCA assay

• Perform Western blot analysis using At3g59230 antibodies

• Quantify band intensity using image analysis software

• Normalize to a loading control (e.g., actin or GAPDH)

For comprehensive stress response characterization, design time-course experiments sampling at multiple timepoints (e.g., 10 min, 1h, 2h, 4h, 24h, 48h, and 96h) after stress application, similar to the approach used in the geomagnetic field response studies .

How can I differentiate between specific and non-specific binding when using At3g59230 antibodies?

Differentiating between specific and non-specific binding is crucial for accurate data interpretation. Implement these strategies:

• Peptide Competition Assay: Pre-incubate the At3g59230 antibody with excess immunizing peptide before application. Specific signals should be significantly reduced or eliminated, while non-specific signals will remain.

• Genetic Controls: Compare results between wild-type plants and At3g59230 knockout/knockdown lines. Specific signals should be absent or reduced in the knockout/knockdown lines.

• Multiple Antibody Approach: Use antibodies targeting different epitopes of At3g59230. Specific signals should be consistent across different antibodies.

• Signal Correlation Analysis: Compare the pattern of At3g59230 protein detection with known expression patterns from transcriptomic data. Correlations between protein and mRNA levels can support signal specificity.

• Mass Spectrometry Validation: For immunoprecipitation experiments, validate pulled-down proteins by mass spectrometry to confirm the presence of At3g59230 and expected interaction partners .

What bioinformatic approaches can help predict the function of At3g59230 based on its protein structure?

To predict At3g59230 function based on its structure, implement these bioinformatic approaches:

• Sequence Homology Analysis: Compare At3g59230 sequence with characterized F-box/LRR proteins using BLAST and multiple sequence alignment tools (MUSCLE, Clustal Omega).

• Domain Prediction and Analysis: Identify functional domains using InterPro, Pfam, and SMART databases. For At3g59230, focus on the F-box domain (protein-protein interaction) and LRR repeats (substrate recognition).

• Structural Modeling: Generate 3D structural models using homology modeling tools (SWISS-MODEL, I-TASSER) or AI-based methods (AlphaFold2). Analyze the structural features of the F-box and LRR domains.

• Protein-Protein Interaction Prediction: Use tools like STRING, MINT, or IntAct to predict interaction partners based on co-expression, experimental data, and database mining.

• Evolutionary Analysis: Perform phylogenetic analysis to identify orthologs in other species with known functions, which can suggest conserved functions.

• Co-expression Network Analysis: Analyze genes co-expressed with At3g59230 across different conditions using tools like ATTED-II to infer functional associations.

Integration of these analyses can provide insights into potential biological roles, subcellular localization, and functional pathways involving At3g59230 .

How do post-translational modifications affect the detection of At3g59230 using specific antibodies?

Post-translational modifications (PTMs) can significantly impact antibody-based detection of At3g59230. Here's how different PTMs might affect detection and strategies to address them:

When investigating PTMs of At3g59230:

• Treat protein samples with modification-specific enzymes (phosphatases, deubiquitinases, etc.) before immunoblotting

• Use PTM-specific antibodies in conjunction with At3g59230 antibodies

• Perform mass spectrometry analysis of immunoprecipitated At3g59230 to map modification sites

• Consider using modification-state specific antibodies if particular PTMs are critical to your research

Understanding the PTM landscape is crucial for correctly interpreting antibody-based detection results and understanding the functional regulation of At3g59230 .

How can CRISPR-Cas9 gene editing be used alongside At3g59230 antibodies to study protein function?

CRISPR-Cas9 gene editing provides powerful complementary approaches when used with At3g59230 antibodies:

• Knockout Validation: Generate At3g59230 knockout lines to serve as negative controls for antibody specificity. The absence of signal in knockout lines confirms antibody specificity.

• Epitope Tagging: Use CRISPR to introduce epitope tags (e.g., FLAG, HA, GFP) into the endogenous At3g59230 locus. This allows detection with highly specific commercial tag antibodies while maintaining native expression levels.

• Domain Function Analysis: Create precise mutations or deletions in specific domains (F-box or LRR repeats) to determine their contribution to protein function, stability, and interactions.

• Promoter Modification: Modify the native promoter to create conditional expression systems, enabling temporal control over At3g59230 expression.

• Functional Complementation: Reintroduce wild-type or mutant versions of At3g59230 into knockout backgrounds to assess functional rescue and structure-function relationships.

When combining CRISPR-Cas9 with antibody-based detection, ensure that your modifications don't alter the epitope recognized by the antibody or affect protein expression and stability in unexpected ways .

What approaches can be used to study At3g59230 protein dynamics during plant development and stress responses?

To study At3g59230 protein dynamics during development and stress responses, implement these approaches:

• Time-course Immunoblotting: Track protein levels at multiple developmental stages or time points after stress treatment using At3g59230 antibodies. Quantify band intensities relative to loading controls.

• Fluorescent Protein Fusions: Generate plants expressing At3g59230-GFP fusions to monitor protein localization and abundance in real-time using confocal microscopy.

• Pulse-Chase Analysis: Label newly synthesized proteins and track At3g59230 stability and turnover rates under different conditions.

• Polysome Profiling: Assess translational regulation by analyzing At3g59230 mRNA association with polysomes during stress responses.

• Protein Degradation Assays: Use cycloheximide chase experiments to determine protein half-life under various conditions.

• Post-translational Modification Profiling: Track changes in phosphorylation, ubiquitination, or other PTMs using modification-specific antibodies or mass spectrometry.

• Tissue-specific Expression Analysis: Combine immunohistochemistry with developmental staging to create protein expression maps across tissues and developmental stages.

Design experiments to include appropriate controls and statistical analyses, with multiple biological replicates to ensure reproducibility. For stress responses, consider analyzing both early (minutes to hours) and late (days) responses to capture the full dynamic range .

How can mass spectrometry complement antibody-based detection of At3g59230 and its interacting partners?

Mass spectrometry (MS) offers powerful complementary approaches to antibody-based detection of At3g59230:

• Protein Identification Confirmation: Validate the identity of antibody-detected bands by excising them from gels and performing MS analysis, confirming that the detected protein is indeed At3g59230.

• Interactome Analysis: Use immunoprecipitation with At3g59230 antibodies followed by MS (IP-MS) to identify the complete set of interacting proteins, revealing unknown partners beyond targeted approaches.

• Post-translational Modification Mapping: Identify and quantify specific PTM sites on At3g59230, providing insights into regulatory mechanisms that antibody detection alone cannot reveal.

• Absolute Quantification: Implement targeted MS approaches (SRM/MRM) to quantify absolute amounts of At3g59230 protein, complementing the relative quantification from immunoblotting.

• Protein Complex Composition: Combine blue native PAGE with MS to characterize native At3g59230-containing complexes and their stoichiometry.

• Spatial Proteomics: Combine laser capture microdissection of specific tissues with MS to determine tissue-specific interactomes.

• Crosslinking MS (XL-MS): Identify direct binding interfaces between At3g59230 and its partners by crosslinking proteins prior to MS analysis.

When integrating MS with antibody-based approaches, design experiments to exploit the strengths of each technique: use antibodies for targeted detection and localization, and MS for unbiased discovery and detailed molecular characterization .

How might understanding At3g59230 function contribute to improving crop stress resilience?

Understanding At3g59230 function could contribute to crop improvement strategies in several ways:

• Stress Response Engineering: If At3g59230 is involved in stress signaling pathways, manipulating its expression or activity could enhance plant resilience to environmental stresses such as drought, salinity, or temperature extremes.

• Protein Turnover Regulation: As an F-box protein likely involved in protein degradation, At3g59230 may regulate the abundance of key stress response factors. Modifying its substrate recognition could optimize stress response pathways.

• Hormone Signaling Modulation: Many F-box proteins participate in hormone signaling (e.g., auxin, jasmonate). If At3g59230 functions in hormone pathways, its modification could enhance growth-stress response balance.

• Developmental Timing Optimization: If At3g59230 regulates developmental transitions, manipulating its function could help synchronize developmental timing with environmental conditions.

• Molecular Breeding Markers: Polymorphisms in At3g59230 correlated with stress resilience could serve as molecular markers for selective breeding programs.

To translate this basic research to crop improvement, researchers would need to:

• Identify At3g59230 orthologs in crop species

• Characterize their function using antibodies and genetic approaches

• Develop targeted modification strategies based on functional insights

• Validate improvements in controlled and field conditions

What is the current understanding of the evolutionary conservation of At3g59230 across plant species?

While the search results don't provide specific information on the evolutionary conservation of At3g59230, F-box/LRR proteins generally show interesting evolutionary patterns:

• Diversification Patterns: F-box proteins often show rapid evolutionary diversification, with expansions in specific lineages reflecting adaptation to different environmental challenges and substrate recognition needs.

• Core F-box Domain Conservation: The F-box domain itself is typically more conserved than the substrate-recognition domains (like LRR), reflecting the conserved interaction with Skp1 proteins across diverse species.

• Functional Conservation vs. Sequence Divergence: Orthologous F-box proteins may maintain similar functions despite sequence divergence, through conservation of critical interaction residues.

To study the evolutionary conservation of At3g59230 specifically:

• Perform phylogenetic analyses using the protein sequence against plant genome databases

• Identify orthologs in key model and crop species

• Compare expression patterns of orthologs under similar conditions

• Test cross-species antibody reactivity to assess structural conservation

• Evaluate functional complementation by expressing orthologs in Arabidopsis At3g59230 mutants

Understanding the evolutionary context could provide insights into the ancestral function of At3g59230 and how it might be specialized in different plant lineages .

How can quantitative proteomics be integrated with At3g59230 antibody-based studies for systems-level understanding?

Integrating quantitative proteomics with At3g59230 antibody-based studies enables a comprehensive systems biology approach:

• Multi-level Analysis Pipeline:

  • Use At3g59230 antibodies for targeted protein detection and localization

  • Apply quantitative proteomics (SILAC, TMT, label-free) for global protein changes

  • Integrate with transcriptomics and metabolomics data

  • Develop computational models of At3g59230-influenced pathways

• Perturbation-Response Mapping:

  • Quantify proteome-wide changes in At3g59230 overexpression/knockout lines

  • Identify direct and indirect effects using time-course analyses

  • Map the propagation of signaling through the proteome

• Protein Interaction Network Dynamics:

  • Combine IP-MS with At3g59230 antibodies at different conditions/timepoints

  • Quantify dynamic changes in interaction partners

  • Correlate with post-translational modification states

• Subcellular Proteome Analysis:

  • Use antibodies to track At3g59230 localization

  • Perform organelle-specific proteomics to identify co-localized proteins

  • Connect spatial and functional relationships

• Multi-species Comparative Proteomics:

  • Apply At3g59230 antibodies across related species if cross-reactive

  • Compare ortholog function and interaction networks

  • Identify conserved and divergent pathway components

Data integration would require advanced computational approaches such as network analysis, machine learning for pattern recognition, and pathway modeling to generate testable hypotheses about At3g59230's role in plant biology .