At1g30730 Antibody

What is the At1g30730 protein and why is it studied in Arabidopsis thaliana?

At1g30730 is a protein encoded by the At1g30730 gene in Arabidopsis thaliana (Mouse-ear cress). According to sequence analysis, the protein contains 158 amino acids with the sequence MKRKSDYVKRPVSRTGLGLILKKLVELEKVEMNWNPYGGRMGEIPSSRTPFPHRGGNLFNIEYIIDWSEAGDNVEKKYLALANEFYRFMTPYVSSNPREAFLNYRDIDIGSSGNSTYEEGKIYGAKYFKDNFERLVDIKTKFDEINFWRNEQSIPVRK . The protein has been identified in studies related to plant signaling pathways, and research suggests it may play a role in plant development or stress responses. It appears in differential expression studies, such as those examining leucine-rich repeat receptor-like kinase1 (RPK1) functions in Arabidopsis .

What types of At1g30730 antibodies are commercially available for research?

Current research antibodies against At1g30730 include:

• Polyclonal antibodies (e.g., CSB-PA219368XA01DOA) raised in rabbits against recombinant Arabidopsis thaliana At1g30730 protein. These are purified using antigen affinity methods and provided in liquid form with preservatives such as 0.03% Proclin 300, 50% Glycerol, and 0.01M PBS (pH 7.4) .

• Combinations of monoclonal antibodies targeting different regions of the protein:

  • X-Q6NNH8-N: Targets N-terminus sequence

  • X-Q6NNH8-C: Targets C-terminus sequence

  • X-Q6NNH8-M: Targets non-terminus (middle) sequence

Each combination contains antibodies raised against multiple synthetic peptides representing the respective region .

What are the validated experimental applications for At1g30730 antibodies?

At1g30730 antibodies have been validated for the following applications:

• Enzyme-Linked Immunosorbent Assay (ELISA): The antibodies demonstrate high-titer binding in ELISA applications, with titers reaching approximately 10,000, which corresponds to detection sensitivity of approximately 1 ng of target protein .

• Western Blotting (WB): The antibodies have been validated for detecting At1g30730 protein in Western blot applications, providing specific identification of the target antigen .

• Expression Analysis: These antibodies can be used in studies examining differential gene expression patterns in Arabidopsis, such as those shown in Table 2 of research publications where At1g30730 exhibited altered expression under specific experimental conditions .

How should At1g30730 antibodies be stored and handled to maintain functionality?

For optimal performance, At1g30730 antibodies should be handled according to these guidelines:

• Storage Temperature: Upon receipt, store at -20°C or -80°C for long-term preservation .

• Avoid Repeated Freeze-Thaw Cycles: Multiple freeze-thaw cycles can damage antibody structure and diminish binding efficiency. Aliquot the antibody solution upon first thaw to minimize freeze-thaw events .

• Working Solution Preparation: When preparing dilutions for experiments, use appropriate buffers as recommended for specific applications (e.g., TBST with 5% BSA or non-fat milk for Western blotting).

• Handling Precautions: Despite being for research use only, follow standard laboratory safety protocols for handling biological materials, including wearing appropriate personal protective equipment.

• Expiration Considerations: Antibody functionality should be validated if used beyond the manufacturer's recommended shelf life, particularly for critical experiments.

How do polyclonal and monoclonal At1g30730 antibodies differ in their detection specificity and experimental applications?

The choice between polyclonal and monoclonal At1g30730 antibodies has significant implications for research outcomes:

Polyclonal At1g30730 Antibodies:

• Recognize multiple epitopes across the target protein, potentially increasing detection sensitivity

• Useful for applications where maximum antigen capture is desired

• May exhibit batch-to-batch variation requiring validation between lots

• Typically provide robust detection in Western blotting and ELISA

• Examples include the rabbit polyclonal antibody CSB-PA219368XA01DOA

Monoclonal At1g30730 Antibodies:

• Target specific epitopes with high precision

• Provide consistent results with minimal batch-to-batch variation

• Particularly valuable for detection of specific protein domains or conformations

• Often used in combinations (as seen with X-Q6NNH8-N/C/M products) to enhance detection while maintaining specificity

• May be engineered to optimize binding characteristics through techniques similar to those used in bispecific antibody development

What are the challenges in validating the specificity of At1g30730 antibodies in Arabidopsis studies?

Validating At1g30730 antibody specificity presents several challenges that researchers must address:

• Cross-reactivity Assessment: At1g30730 may share sequence homology with other Arabidopsis proteins, necessitating thorough validation through:

  • Western blot analysis using recombinant At1g30730 protein as a positive control

  • Testing against known knockout/knockdown lines where the At1g30730 gene has been silenced

  • Peptide competition assays to confirm binding specificity

• Expression Level Variability: At1g30730 expression varies across tissues and under different experimental conditions, as evidenced in differential expression studies . This variability requires:

  • Tissue-specific validation

  • Calibration of detection methods across different expression levels

  • Inclusion of appropriate loading controls

• Post-translational Modifications: Potential modifications may affect epitope accessibility, requiring:

  • Multiple antibodies targeting different regions (similar to the N/C/M-terminal approach )

  • Validation under conditions that may induce different modifications

• Technical Validation Standards: Include:

  • Signal-to-noise ratio determination

  • Limit of detection establishment

  • Reproducibility across multiple experimental replicates

While similar to validation processes for other plant protein antibodies, these challenges are particularly relevant for At1g30730 due to its expression patterns in Arabidopsis signaling studies.

How can At1g30730 antibodies be effectively used in multi-parameter protein studies in plant systems?

Leveraging At1g30730 antibodies in multi-parameter studies requires strategic experimental design:

Co-localization Studies:

• Combine At1g30730 antibodies with antibodies against known interacting proteins or organelle markers

• Use secondary antibodies with distinct fluorophores that have minimal spectral overlap

• Include appropriate controls to account for potential antibody cross-reactivity

Protein Complex Analysis:

• For co-immunoprecipitation studies, optimize buffer conditions to maintain protein interactions while ensuring efficient At1g30730 capture

• Consider the use of crosslinking approaches to stabilize transient interactions

• Validate results with reciprocal co-immunoprecipitation using antibodies against putative interacting partners

Sequential Immunodetection:
When performing multiple rounds of immunodetection on the same membrane:

• Document complete stripping of previous antibodies before reprobing

• Consider the order of detection based on antibody sensitivity (typically detecting lower abundance proteins first)

• Validate that the stripping process does not affect the integrity of the immobilized proteins

Data Integration Table Example:

What are the most effective strategies for using At1g30730 antibodies in studies of plant stress responses?

When investigating plant stress responses with At1g30730 antibodies, consider these evidence-based approaches:

• Temporal Expression Profiling:

  • Collect samples at multiple time points post-stress treatment

  • Use Western blotting with At1g30730 antibodies to track protein level changes

  • Correlate protein expression with transcriptional changes using parallel RT-qPCR analysis

• Spatial Distribution Analysis:

  • Employ immunohistochemistry or immunofluorescence using fixed tissue sections

  • Compare At1g30730 protein localization across different tissues under stress vs. control conditions

  • Validate immunolocalization results with GFP-fusion protein approaches if available

• Functional Analysis Through Protein Interaction Studies:

  • Use At1g30730 antibodies for immunoprecipitation followed by mass spectrometry to identify stress-induced interaction partners

  • Compare interactomes under normal vs. stress conditions

  • Validate key interactions through techniques like bimolecular fluorescence complementation

• Modification Status Assessment:

  • Investigate post-translational modifications under stress conditions using phospho-specific antibodies (if available)

  • Compare migration patterns in Western blots under different stress treatments

  • Consider 2D gel electrophoresis followed by immunoblotting to resolve modified forms

Research indicates that At1g30730 expression changes in response to certain experimental conditions, as shown in Table 2 of a study examining differential gene expression patterns . This suggests potential involvement in stress response pathways that warrant further investigation using these strategies.

What optimization steps are recommended for Western blotting with At1g30730 antibodies?

Achieving optimal Western blot results with At1g30730 antibodies requires systematic optimization:

Sample Preparation:

• Extract proteins using buffer systems that preserve At1g30730 integrity (typically containing protease inhibitors and phosphatase inhibitors if phosphorylation is relevant)

• Determine optimal protein loading amounts (typically 20-50 μg total protein per lane for standard plant extracts)

• Include appropriate positive controls (e.g., recombinant At1g30730 protein) and negative controls (e.g., extracts from knockout lines if available)

Electrophoresis and Transfer Parameters:

• Use 12-15% SDS-PAGE gels for optimal resolution of the 158 amino acid At1g30730 protein

• Consider gradient gels when analyzing potential protein complexes

• Optimize transfer conditions based on protein size (typically 100V for 1 hour for proteins of this size)

Antibody Incubation:

• Determine optimal primary antibody dilution through titration experiments (starting with manufacturer recommendations, typically 1:1000 for polyclonal antibodies)

• Test different blocking agents (5% BSA vs. 5% non-fat milk) to minimize background

• Optimize incubation temperature and duration (4°C overnight vs. room temperature for 1-2 hours)

Detection Optimization:

• Select detection systems based on expected expression levels (chemiluminescence for standard detection, fluorescent secondary antibodies for quantitative analysis)

• Optimize exposure times to prevent signal saturation

• Consider signal amplification systems for low abundance detection

Quantitative Analysis:

• Use housekeeping proteins appropriate for the experimental context as loading controls

• Apply densitometry analysis with appropriate normalization

• Ensure analysis is performed within the linear range of detection

How can immunoprecipitation protocols be optimized for At1g30730 protein complex studies?

For successful immunoprecipitation of At1g30730 and associated protein complexes:

Pre-immunoprecipitation Considerations:

• Determine appropriate lysis buffer composition based on subcellular localization and interaction stability

• Optimize cell/tissue disruption methods to maintain protein complex integrity

• Establish appropriate protein concentration (typically 1-5 mg/ml) and volume for efficient immunoprecipitation

Antibody Selection and Coupling:

• Compare the efficiency of different At1g30730 antibodies for immunoprecipitation

• Consider using a combination of antibodies targeting different epitopes to maximize capture

• For large-scale studies, covalently couple antibodies to solid support (e.g., using crosslinkers such as BS3 or DMP)

Immunoprecipitation Protocol Optimization:

• Determine optimal antibody-to-protein ratio through titration experiments

• Optimize incubation conditions (4°C overnight with gentle rotation is typically effective)

• Establish appropriate washing stringency to remove non-specific interactions while preserving specific complexes

Validation and Analysis Approaches:

• Confirm successful precipitation using Western blotting with a portion of the immunoprecipitated material

• For interactome studies, process samples for mass spectrometry following established protocols

• Include appropriate controls including:

  • IgG control (same species as the primary antibody)

  • Input sample (pre-immunoprecipitation lysate)

  • When possible, immunoprecipitation from knockout/knockdown lines

A typical immunoprecipitation workflow involves:

• Lysate preparation (1-2 hours)

• Pre-clearing with control IgG and protein A/G beads (1 hour)

• Antibody incubation (overnight)

• Bead capture (1-2 hours)

• Washing steps (1-2 hours)

• Elution and analysis (varies by downstream application)

What are the critical considerations for using At1g30730 antibodies in immunohistochemistry of plant tissues?

Successful immunohistochemistry with At1g30730 antibodies requires attention to these critical parameters:

Tissue Preparation:

• Optimize fixation methods (4% paraformaldehyde is standard, but test multiple fixatives)

• Determine appropriate section thickness (typically 5-10 μm for plant tissues)

• Consider antigen retrieval methods if epitope masking is suspected during fixation

Antibody Validation for IHC:

• Test antibody specificity on known At1g30730-expressing tissues

• Include appropriate negative controls (pre-immune serum, antibody pre-absorption with immunizing peptide)

• Compare staining patterns with in situ hybridization or reporter gene expression if available

Protocol Optimization:

• Determine effective blocking conditions to minimize non-specific binding

• Establish optimal primary antibody dilution (typically starting at 1:100-1:500 for polyclonal antibodies)

• Optimize incubation conditions (temperature, duration, humidity)

• Select appropriate detection systems based on required sensitivity and analysis methods

Counterstaining and Visualization:

• Choose counterstains that provide tissue context without interfering with specific signal

• For co-localization studies, ensure secondary antibodies have minimal spectral overlap

• Establish imaging parameters that capture specific signal while minimizing background

Quantitative Analysis Approaches:

• Develop consistent scoring methods for signal intensity

• Use digital image analysis software with appropriate thresholding

• Include internal standardization for cross-sample comparisons

Given that At1g30730 has been studied in the context of plant signaling pathways, pay particular attention to its potential co-localization with known signaling components and membrane structures during immunohistochemical analysis.

How should researchers troubleshoot non-specific binding when using At1g30730 antibodies?

When encountering non-specific binding with At1g30730 antibodies, implement this systematic troubleshooting approach:

Problem Diagnosis:

• Characterize the pattern of non-specific binding:

  • Multiple unexpected bands in Western blots

  • Diffuse or universal staining in immunohistochemistry

  • High background in ELISA

• Evaluate potential causes through controlled experiments:

  • Antibody quality issues (test new lot if available)

  • Sample preparation problems (optimize extraction/fixation)

  • Detection system sensitivity (adjust exposure/development time)

Optimization Strategies:

Experimental Validation Approaches:

• Peptide competition assay: Pre-incubate antibody with immunizing peptide before application

• Knockout/knockdown control: Test antibody on tissues/cells with reduced/absent At1g30730 expression

• Epitope-tagged protein expression: Compare antibody binding pattern with anti-tag antibody detection

Advanced Refinement Techniques:

• Antibody purification through antigen-affinity chromatography

• Cross-adsorption against related proteins to remove cross-reactive antibodies

• Isotype-specific secondary antibody selection for reduced background

If non-specific binding persists, consider testing alternative antibodies targeting different epitopes of At1g30730, as molecular engineering approaches have demonstrated that binding specificity can be significantly improved through epitope selection optimization .

How can researchers correctly interpret At1g30730 antibody data in the context of plant development studies?

Proper interpretation of At1g30730 antibody data requires contextual analysis and rigorous controls:

Baseline Expression Context:

• Establish normal At1g30730 expression patterns across:

  • Different developmental stages

  • Various tissue types

  • Standard growth conditions

• Quantify typical expression levels in reference tissues to enable comparative analysis

Experimental Design Considerations:

• Include appropriate sampling intervals to capture developmental dynamics

• Establish statistical power through biological and technical replicates

• Design time-course experiments to correlate At1g30730 expression with developmental transitions

Data Interpretation Framework:

• Compare protein expression (antibody-based detection) with transcriptional data (RT-qPCR, RNA-seq)

• Analyze subcellular localization changes during development

• Correlate At1g30730 expression with known developmental markers

Contextual Analysis Example:
Previous research has shown differential expression patterns for At1g30730 in certain experimental conditions . When interpreting experimental data:

• Upregulation might suggest involvement in specific developmental pathways

• Changes in subcellular localization may indicate activation/inactivation

• Co-expression with known developmental regulators may suggest functional relationships

Causal Relationship Assessment:

• Distinguish correlation from causation through functional studies

• Consider genetic approaches (knockout/knockdown/overexpression) to validate antibody-derived observations

• Use pharmacological interventions when appropriate to disrupt suspected pathways

Integration with Broader Knowledge:

• Compare findings with existing literature on At1g30730 and related proteins

• Consider evolutionary conservation of expression patterns across related species

• Develop testable hypotheses based on observed expression patterns

What are the best practices for using At1g30730 antibodies in quantitative proteomics studies?

Incorporating At1g30730 antibodies into quantitative proteomics requires methodological rigor:

Sample Preparation Considerations:

• Standardize protein extraction protocols to ensure consistent recovery

• Include spike-in standards for absolute quantification when necessary

• Consider subcellular fractionation to enrich for At1g30730-containing compartments

Antibody-Based Enrichment Strategies:

• For targeted proteomics, optimize immunoprecipitation conditions for At1g30730 and interacting partners

• Consider sequential immunoprecipitation to identify specific interaction complexes

• Validate enrichment efficiency through Western blotting before mass spectrometry analysis

Quantitative Analysis Approaches:

Validation Requirements:

• Confirm mass spectrometry identifications with orthogonal methods (Western blotting, immunofluorescence)

• Validate protein-protein interactions through reciprocal co-immunoprecipitation

• Correlate protein abundance changes with functional outcomes

Data Integration Approaches:

• Map quantitative changes to protein interaction networks

• Correlate protein abundance with post-translational modifications

• Integrate proteomics data with transcriptomics and metabolomics for systems-level analysis

Similar approaches have been successfully employed in studies of plant signaling pathways, where quantitative proteomics revealed dynamic changes in protein complexes during signal transduction .

How can researchers evaluate and compare the performance of different commercially available At1g30730 antibodies?

Systematic evaluation of different At1g30730 antibodies requires standardized comparative analysis:

Initial Assessment Parameters:

• Antibody characteristics documentation:

  • Target epitope location and sequence

  • Host species and antibody type (polyclonal vs. monoclonal)

  • Production method and purification approach

  • Validated applications according to manufacturer

• Validation documentation review:

  • Specificity data provided by manufacturer

  • Published literature using the antibody

  • Independent validation studies if available

Comparative Performance Testing:

Standardized Testing Protocol Example:

• Prepare identical samples from:

  • Wild-type Arabidopsis (positive control)

  • At1g30730 knockout/knockdown lines if available (negative control)

  • Recombinant At1g30730 protein (reference standard)

• Process all samples in parallel using standardized protocols

• Test each antibody under identical conditions:

  • Same blocking reagents and concentrations

  • Consistent incubation times and temperatures

  • Identical detection systems and exposure times

• Quantitatively evaluate performance metrics:

  • Signal-to-background ratio

  • Detection limit

  • Dynamic range

  • Cross-reactivity profile

Documentation and Selection Criteria:

• Create a comprehensive comparison table including all testing parameters

• Weight performance criteria according to experimental requirements

• Consider cost-effectiveness for long-term experimental planning

• Document selected antibody characteristics for methods sections in publications

When comparing commercial antibodies like CSB-PA219368XA01DOA with other options such as the combination monoclonal sets (X-Q6NNH8-N/C/M) , prioritize the performance metrics most relevant to your specific experimental applications.

How might emerging antibody engineering technologies improve At1g30730 antibody performance in plant research?

Advanced antibody engineering approaches offer significant potential improvements for At1g30730 research:

Current Limitations and Engineering Solutions:

• Specificity Enhancement:

  • Implementation of phage display technologies to select high-specificity binding domains

  • Development of recombinant antibodies with optimized complementarity-determining regions (CDRs)

  • Application of deep mutational scanning to identify variants with improved specificity

• Sensitivity Improvement:

  • Engineering of bispecific antibodies targeting multiple At1g30730 epitopes simultaneously

  • Development of antibody fragments with enhanced tissue penetration

  • Creation of antibody-enzyme conjugates for signal amplification

• Functional Capability Expansion:

  • Integration of pH-dependent binding properties similar to "sweeping antibodies"

  • Development of conformation-specific antibodies to distinguish active/inactive states

  • Creation of intrabodies for in vivo tracking of At1g30730

Emerging Technologies with Potential Application:

Implementation Considerations:

• Validation requirements for engineered antibodies in plant research

• Cost-effectiveness compared to traditional antibody production

• Technical expertise required for implementation in standard plant biology laboratories

The development of bispecific antibodies with enhanced specificity and functionality, as described in recent research , represents a particularly promising approach for improving At1g30730 detection in complex plant samples.

What role might At1g30730 antibodies play in advancing our understanding of plant stress signaling networks?

At1g30730 antibodies could significantly advance plant stress signaling research through these approaches:

Signaling Network Mapping:

• Use At1g30730 antibodies in proximity labeling approaches (BioID, APEX) to identify proteins in spatial proximity during stress responses

• Apply quantitative immunoprecipitation followed by mass spectrometry to characterize dynamic interaction networks

• Employ antibody-based ChIP-seq to identify potential DNA-binding activities if relevant

Spatiotemporal Dynamics Analysis:

• Track At1g30730 protein relocalization during stress responses using immunofluorescence

• Quantify protein abundance changes across tissues and time points using quantitative Western blotting

• Investigate post-translational modifications using modification-specific antibodies if available

Functional Studies:

• Use antibodies to inhibit protein function in cell-free systems or through microinjection

• Develop antibody-based biosensors to track protein conformation changes during signaling

• Apply antibodies in protein-array technologies to identify novel interaction partners

Integration with Existing Knowledge:
Research has shown that At1g30730 expression can be altered in experimental conditions related to plant signaling studies . Based on this foundation, antibody-based approaches could:

• Determine whether transcriptional changes correlate with protein-level changes

• Identify post-translational modifications that may regulate protein function

• Elucidate protein-protein interactions that may connect At1g30730 to known signaling pathways

Methodological Innovations:

• Application of single-molecule imaging techniques using fluorescently labeled antibodies or antibody fragments

• Development of antibody-based optogenetic tools for controlling protein function

• Integration of antibody-based detection with spatial transcriptomics for comprehensive pathway analysis

Similar spatiotemporal analysis approaches have successfully identified key transcription factors in plant stress responses, as seen in recent studies , and could be applied to understand the potential role of At1g30730 in these pathways.

How can computational approaches enhance the design and application of At1g30730 antibodies in plant research?

Computational methods offer powerful strategies to optimize At1g30730 antibody research:

Epitope Prediction and Antibody Design:

• Application of machine learning algorithms to predict optimal epitopes based on:

  • Protein structure prediction (if 3D structure is unknown)

  • Surface accessibility analysis

  • Evolutionary conservation assessment

  • Post-translational modification site prediction

• In silico antibody design approaches:

  • Computational modeling of antibody-antigen interactions

  • Optimization of binding affinity through virtual mutations

  • Design of multi-epitope recognition strategies

Active Learning for Improved Antibody Selection:
Recent research has demonstrated that active learning strategies can significantly improve antibody-antigen binding prediction . Applied to At1g30730:

• Establish initial small-scale experimental dataset of antibody binding characteristics

• Apply active learning algorithms to predict optimal antibody candidates

• Iteratively test and refine predictions to identify optimal antibodies with minimal experimental investment

Data Integration and Analysis Enhancement:

Implementation Pathway:

• Establish collaborations between plant biologists and computational scientists

• Develop plant-specific datasets to train machine learning models

• Validate computational predictions through targeted experimental approaches

• Iterate between computational prediction and experimental validation

Research has shown that active learning strategies can reduce the number of required experimental variants by up to 35% and speed up the learning process by 28 steps compared to random approaches . This efficiency gain could significantly accelerate At1g30730 research while reducing experimental costs.