At3g62230 Antibody

What is At3g62230 Antibody and what are its fundamental properties?

At3g62230 Antibody (Product Code: CSB-PA885514XA01DOA) is a polyclonal, antigen-affinity purified antibody raised in rabbits against recombinant Arabidopsis thaliana At3g62230 protein. This antibody specifically recognizes the At3g62230 protein in Arabidopsis thaliana (Mouse-ear cress). It is supplied in liquid form containing 50% glycerol, 0.01M PBS (pH 7.4), and 0.03% Proclin 300 as a preservative. The antibody has been validated for ELISA and Western blot applications, making it suitable for detecting and quantifying At3g62230 protein in research contexts .

What are the recommended storage and handling conditions for At3g62230 Antibody?

For optimal preservation of antibody activity and functionality, At3g62230 Antibody should be stored at either -20°C or -80°C immediately upon receipt. The manufacturer specifically advises avoiding repeated freeze-thaw cycles as these can significantly compromise antibody stability and activity. Best practices include aliquoting the antibody into smaller volumes based on experimental needs to minimize freeze-thaw cycles, and allowing aliquots to reach room temperature before use. The antibody is provided in a storage buffer containing 50% glycerol, which helps maintain stability during freeze-thaw transitions .

How should researchers validate the specificity of At3g62230 Antibody before experimental use?

Rigorous validation of At3g62230 Antibody specificity is essential for ensuring reliable experimental results. A comprehensive validation approach should include:

• Testing the antibody in Arabidopsis thaliana knockout or knockdown lines lacking the At3g62230 gene (T-DNA insertion lines or CRISPR-edited plants)

• Performing peptide competition assays by pre-incubating the antibody with excess immunizing peptide

• Verifying that detected bands in Western blot match the predicted molecular weight of At3g62230 protein

• Using recombinant At3g62230 protein as a positive control alongside related proteins as negative controls

• Comparing antibody detection with orthogonal methods such as RNA expression analysis or mass spectrometry

These validation steps help establish confidence in antibody specificity, which is crucial for meaningful interpretation of experimental results, particularly given the potential for cross-reactivity with related plant proteins .

How should At3g62230 Antibody be optimized for Western blot applications?

Optimizing At3g62230 Antibody for Western blot applications requires systematic adjustment of multiple parameters to achieve the best signal-to-noise ratio. Key optimization steps include:

• Determining the optimal antibody dilution through a titration series (typically starting with 1:500 to 1:5000)

• Testing different blocking agents (5% non-fat milk, 3-5% BSA, or commercial blocking buffers)

• Optimizing incubation conditions (typically overnight at 4°C or 1-2 hours at room temperature)

• Refining sample preparation protocols to ensure efficient protein extraction while preserving epitope integrity

• Implementing appropriate controls (positive samples containing At3g62230 protein and negative controls)

The following table summarizes recommended optimization parameters:

Systematic optimization of these parameters will help researchers achieve the most sensitive and specific detection of At3g62230 protein .

What strategies can improve detection sensitivity when working with low-abundance At3g62230 protein?

When working with low-abundance At3g62230 protein, researchers can implement several strategies to enhance detection sensitivity:

• Sample enrichment techniques such as immunoprecipitation to concentrate the target protein before analysis

• Utilizing enhanced chemiluminescent (ECL) substrates specifically designed for high-sensitivity detection

• Implementing signal amplification methods such as tyramide signal amplification (TSA) or polymer-based detection systems

• Extending primary antibody incubation times (up to 48 hours at 4°C) to maximize binding opportunities

• Optimizing protein extraction methods to improve solubilization and recovery of At3g62230 protein

• Using low-fluorescence or specialized PVDF membranes designed for enhanced protein binding and reduced background

Additionally, researchers can explore alternative detection platforms such as capillary Western systems or automated Western blot processors that often provide improved sensitivity through standardized procedures and specialized detection chemistries. When implementing these approaches, it remains essential to include appropriate controls to validate the specificity of detected signals, particularly when working at the limits of detection .

How can researchers design experiments to study At3g62230 protein interactions using the antibody?

Designing experiments to study At3g62230 protein interactions requires strategic application of the antibody in various interaction detection methodologies:

• Co-immunoprecipitation (Co-IP): Use At3g62230 Antibody to precipitate the target protein along with its binding partners from plant lysates, followed by mass spectrometry identification or Western blot detection of co-precipitated proteins.

• Proximity Ligation Assay (PLA): Combine At3g62230 Antibody with antibodies against suspected interaction partners to visualize protein-protein interactions in situ with single-molecule resolution.

• Chromatin Immunoprecipitation (ChIP): If At3g62230 is involved in transcriptional regulation, use the antibody to identify DNA sequences associated with the protein.

• Bimolecular Fluorescence Complementation (BiFC): Although this technique doesn't directly use the antibody, it can complement antibody-based approaches by confirming interactions identified through Co-IP.

• Pull-down validation: Use recombinant At3g62230 protein to validate interactions identified through other methods, with the antibody serving to detect bound proteins.

A comprehensive experimental design should include:

• Appropriate negative controls (IgG controls, competing peptides)

• Reciprocal experiments where possible (precipitating with antibodies against suspected partners)

• Validation through orthogonal methods

• Functional assays to confirm the biological relevance of identified interactions

These approaches can help researchers map the interactome of At3g62230 protein and understand its role in plant biological processes .

What are common issues encountered with At3g62230 Antibody in Western blot and how can they be resolved?

Researchers commonly encounter several challenges when using At3g62230 Antibody in Western blot applications. These issues and their potential solutions include:

• High background signal:

  • Increase blocking time or concentration (try 5% BSA instead of milk)

  • Reduce primary antibody concentration

  • Add 0.1-0.3% Tween-20 to washing buffer

  • Increase number and duration of washing steps

• Weak or absent signal:

  • Optimize protein extraction to preserve epitope integrity

  • Increase antibody concentration or incubation time

  • Ensure sample hasn't degraded during preparation

  • Try alternative membrane types (PVDF often performs better than nitrocellulose for some antibodies)

  • Use enhanced sensitivity detection systems

• Multiple bands or unexpected band sizes:

  • Verify if bands represent degradation products, post-translational modifications, or splice variants

  • Include peptide competition controls to identify specific versus non-specific bands

  • Test samples from knockout plants as negative controls

  • Optimize sample preparation to reduce proteolysis

• Inconsistent results between experiments:

  • Standardize all aspects of the protocol (sample preparation, blocking, antibody dilutions)

  • Prepare fresh working solutions for each experiment

  • Include internal loading controls

  • Document lot numbers and prepare larger antibody aliquots to minimize variation

• Edge effects or uneven signal across the membrane:

  • Ensure adequate volume of antibody solution to completely cover the membrane

  • Agitate gently during incubations

  • Use incubation trays or heat-sealed bags to prevent membrane drying

Systematic troubleshooting by modifying one parameter at a time will help identify and address the specific causes of problems in each experimental system .

How can advanced imaging technologies enhance the application of At3g62230 Antibody in cellular localization studies?

Advanced imaging technologies can significantly enhance the application of At3g62230 Antibody for cellular localization studies, providing superior resolution and quantitative data:

• Super-resolution microscopy techniques:

  • Structured Illumination Microscopy (SIM) can achieve resolution of ~100 nm

  • Stimulated Emission Depletion (STED) microscopy offers resolution down to 30-80 nm

  • Single Molecule Localization Microscopy (SMLM) methods like PALM or STORM can reach 10-20 nm resolution

  • These techniques enable precise localization of At3g62230 protein relative to subcellular structures

• Multi-color confocal microscopy:

  • Simultaneous imaging of At3g62230 (using labeled secondary antibodies) alongside organelle markers

  • Spectral unmixing capabilities to resolve signals with overlapping emission spectra

  • Live-cell compatible imaging strategies for dynamic localization studies

• 3D imaging approaches:

  • Z-stack acquisition and 3D reconstruction to visualize spatial distribution

  • Light sheet microscopy for reduced photobleaching and phototoxicity

  • Volume rendering for comprehensive visualization of protein distribution

• Quantitative image analysis:

  • Colocalization analysis using Pearson's or Mander's coefficients

  • Intensity correlation analysis

  • Distance-based approaches for proximity measurements

  • Machine learning algorithms for pattern recognition and classification

• Correlative Light and Electron Microscopy (CLEM):

  • Combines immunofluorescence detection of At3g62230 with ultrastructural context

  • Particularly valuable for precise localization within complex organelles

These advanced imaging approaches enable researchers to not only visualize the subcellular distribution of At3g62230 protein but also quantify its abundance and characterize its dynamic behavior under different experimental conditions .

How can researchers evaluate and enhance the thermal stability of At3g62230 Antibody for long-term studies?

Evaluating and enhancing the thermal stability of At3g62230 Antibody is crucial for ensuring consistent performance in long-term studies. Researchers can employ the following approaches:

• Stability assessment methodologies:

  • Differential Scanning Fluorimetry (DSF) to monitor thermal unfolding profiles

  • Size Exclusion Chromatography (SEC) to detect aggregation or fragmentation

  • Enzyme-Linked Immunosorbent Assay (ELISA) to measure functional binding activity after thermal stress

  • Periodic Western blot testing against standard samples to track performance over time

• Stability-enhancing storage conditions:

  • Store in small aliquots (20-50 μl) at -80°C to minimize freeze-thaw cycles

  • Add stabilizing agents like trehalose (0.5-1%) or additional glycerol if not already present

  • Consider lyophilization for very long-term storage needs

  • Maintain strict temperature records and avoid temperature fluctuations

• Buffer optimization strategies:

  • Adjust pH to optimal range (typically pH 7.2-7.5)

  • Add carrier proteins like BSA (0.1-1%) if not already in formulation

  • Test addition of antioxidants like 1-5 mM EDTA or 1 mM sodium azide

  • Evaluate commercial antibody stabilization solutions

• Handling practices to preserve stability:

  • Minimize exposure to light and heat during experiments

  • Use non-stick tubes for storage to prevent adsorptive loss

  • Avoid vortexing; mix by gentle inversion

  • Centrifuge briefly before use to collect contents after thawing

The following table summarizes stability assessment approaches and their applications:

Implementing these approaches allows researchers to systematically monitor and enhance antibody stability, ensuring reliable performance throughout extended experimental timelines .

How does At3g62230 protein function in plant signaling pathways based on current research?

The At3g62230 protein (UniProt: Q9M1Q1) is characterized as an F-box/kelch-repeat protein, functioning as a substrate recognition component in SCF (Skp1-Cullin-F-box) E3 ubiquitin ligase complexes. These complexes play crucial roles in protein ubiquitination and subsequent degradation via the 26S proteasome pathway, representing a fundamental regulatory mechanism in plant signaling.

Current research indicates that At3g62230 is involved in several signaling pathways:

The At3g62230 Antibody provides researchers with a valuable tool to investigate these functions through protein detection, localization studies, and interaction analyses, contributing to our understanding of plant molecular signaling networks .

What in silico approaches can complement experimental work with At3g62230 Antibody?

In silico approaches can significantly enhance experimental work with At3g62230 Antibody, providing computational frameworks that both guide and complement laboratory investigations:

• Structural modeling and analysis:

  • Homology modeling of At3g62230 protein structure based on related F-box/kelch-repeat proteins

  • Molecular docking simulations to predict interactions with potential substrates or partners

  • Molecular dynamics simulations to analyze protein flexibility and conformational changes

  • These approaches can inform epitope accessibility for antibody binding

• Epitope prediction and antibody engineering:

  • Computational prediction of immunogenic epitopes within At3g62230 protein

  • Antibody modeling to understand paratope-epitope interactions

  • In silico affinity maturation to design improved variants with enhanced specificity

  • These methods can help researchers interpret and potentially improve antibody performance

• Network analysis and systems biology:

  • Prediction of functional protein-protein interaction networks involving At3g62230

  • Integration with transcriptomic and proteomic datasets to identify co-regulated genes/proteins

  • Pathway enrichment analysis to contextualize At3g62230 within broader biological processes

  • These approaches guide hypothesis generation for experimental validation

• Experimental design optimization:

  • Statistical modeling to determine optimal experimental conditions

  • Design of Experiments (DoE) approaches to efficiently explore parameter spaces

  • Power analysis to determine appropriate sample sizes for meaningful results

  • These methods enhance experimental efficiency and statistical robustness

• Image analysis for microscopy applications:

  • Automated quantification of immunofluorescence signals

  • Colocalization analysis algorithms for multi-channel imaging

  • Machine learning approaches for pattern recognition in complex tissues

  • These tools extract quantitative data from antibody-based imaging

The integration of these computational approaches with experimental work using At3g62230 Antibody creates a powerful research strategy that maximizes data interpretation while minimizing experimental iterations .

How can researchers apply advanced protein characterization techniques in conjunction with At3g62230 Antibody?

Researchers can employ several advanced protein characterization techniques in conjunction with At3g62230 Antibody to gain deeper insights into protein function, interactions, and dynamics:

• Surface Plasmon Resonance (SPR) and Bio-Layer Interferometry (BLI):

  • Quantify binding kinetics and affinity constants between At3g62230 and interaction partners

  • Measure association (ka) and dissociation (kd) rates and equilibrium dissociation constants (KD)

  • Compare binding properties across different experimental conditions or mutations

  • These techniques require immobilizing either At3g62230 protein or its partners, with the antibody potentially serving as a capture reagent

• Hydrogen-Deuterium Exchange Mass Spectrometry (HDX-MS):

  • Map regions of At3g62230 involved in protein-protein interactions

  • Identify conformational changes upon substrate binding

  • Characterize structural dynamics in different cellular states

  • The antibody can be used to immunoprecipitate At3g62230 protein complexes prior to HDX-MS analysis

• Cryo-Electron Microscopy and X-ray Crystallography:

  • Determine high-resolution structures of At3g62230 protein alone or in complexes

  • Visualize the structural basis for substrate recognition and binding

  • The antibody might aid in complex stabilization or crystallization

  • Antibody-based purification can provide sufficient quantities of pure protein for structural studies

• Cross-linking Mass Spectrometry (XL-MS):

  • Identify direct contact points between At3g62230 and its binding partners

  • Map the topography of protein complexes

  • The antibody can be used for immunoprecipitation of cross-linked complexes

• Single-Molecule Techniques:

  • Single-molecule FRET to monitor conformational changes

  • Optical tweezers or atomic force microscopy to measure mechanical properties

  • These approaches provide insights into protein dynamics invisible to ensemble measurements

• Proximity Labeling Methods:

  • BioID or APEX2 fusion proteins to identify the proximal interactome of At3g62230

  • The antibody serves to validate proximity labeling results

These advanced characterization techniques, when combined with traditional antibody-based approaches, provide a comprehensive toolset for investigating At3g62230 protein function at multiple levels of resolution, from atomic structure to cellular context .