At3g58820 Antibody

What is the At3g13820 antibody and what target does it recognize?

The At3g13820 antibody is a rabbit polyclonal antibody that specifically recognizes the At3g13820 protein (also known as F-box protein At3g13820) from Arabidopsis thaliana (Mouse-ear cress). This antibody targets a protein with a molecular weight of approximately 47,623 Da and is developed through antigen-affinity purification methods . The antibody is designed for research applications requiring specific detection of this F-box protein, which plays roles in protein-protein interactions and potentially in ubiquitin-mediated protein degradation pathways in plants. When designing experiments, researchers should account for the polyclonal nature of this antibody, which means it recognizes multiple epitopes on the target protein, potentially increasing sensitivity but requiring careful validation to ensure specificity.

What are the recommended applications for the At3g13820 antibody?

The At3g13820 antibody is primarily designed for research applications in plant molecular biology. While specific application data for this particular antibody is limited in the search results, general antibody applications would include Western blotting, immunoprecipitation, immunohistochemistry, and potentially flow cytometry with appropriate validation . When planning experiments, researchers should first validate the antibody's performance in their specific application and model system. For instance, when considering flow cytometry applications, researchers should employ validation approaches such as genetic knockdowns, comparison with orthogonal methods, or using cell lines with known expression levels of the target protein to confirm specificity and optimal working conditions for the antibody .

How should researchers properly store and handle the At3g13820 antibody?

The At3g13820 antibody should be stored at -20°C or -80°C upon receipt to maintain its functionality and specificity . The antibody is typically provided in a liquid format containing preservatives such as 0.03% Proclin 300 and stabilizers including 50% glycerol and 0.01M PBS at pH 7.4 . To ensure optimal performance, researchers should avoid repeated freeze-thaw cycles which can degrade antibody performance. If small volumes of antibody become entrapped in the seal of the product vial during shipment or storage, a brief centrifugation on a tabletop centrifuge is recommended to dislodge the liquid . Additionally, researchers should consider preparing small aliquots for single-use applications to minimize exposure to adverse conditions and prevent contamination that could compromise experimental results.

What controls should be included when using the At3g13820 antibody in experiments?

When using the At3g13820 antibody, multiple controls are essential to ensure experimental validity:

• Positive control: Samples known to express the At3g13820 protein, such as wild-type Arabidopsis thaliana tissues

• Negative control: Samples lacking the target protein, such as:

  • At3g13820 knockout/knockdown lines

  • Non-plant samples or plant species that don't express homologous proteins

• Technical controls:

  • Secondary antibody-only control to detect non-specific binding

  • Isotype control (rabbit IgG) to assess background signal

  • Blocking peptide competition assay to confirm specificity

Incorporating these controls helps identify false positives and negatives, particularly important when validating antibody selectivity in flow cytometry or other applications . For quantitative experiments, researchers should also include standard curves using recombinant At3g13820 protein when possible, to ensure signal linearity within the working range of the assay.

What validation strategies should be employed to confirm At3g13820 antibody specificity?

Rigorous validation of the At3g13820 antibody requires multiple complementary approaches:

As noted in search result , antibody validation for applications like flow cytometry often requires combining overexpression with orthogonal approaches and cell treatments to establish reliability. Researchers should be aware that factors such as inefficient knockdown at RNA or protein levels and off-target RNAi activities can complicate interpretation . For proteins critical to cell survival, only partial knockdown may be achievable, making validation more challenging. The validation approach should be adapted according to the specific experimental context, target characteristics, and available resources.

How can researchers troubleshoot inconsistent results when using the At3g13820 antibody?

When encountering inconsistent results with the At3g13820 antibody, researchers should systematically investigate several potential factors:

• Sample preparation issues:

  • Ensure proper protein extraction methods for plant tissues

  • Verify protein denaturation conditions are appropriate

  • Check for proteolytic degradation with protease inhibitors

• Experimental conditions optimization:

  • Titrate antibody concentration

  • Modify blocking conditions to reduce background

  • Adjust incubation times and temperatures

  • Test different detection systems

• Target protein biology:

  • Consider post-translational modifications affecting epitope recognition

  • Evaluate protein expression variability under different growth conditions

  • Assess protein localization changes that might affect accessibility

• Antibody quality:

  • Test different lots of the antibody

  • Confirm proper storage conditions were maintained

  • Consider antibody degradation over time

For flow cytometry applications specifically, inconsistent results might stem from cell preparation methods, fixation protocols, or buffer compositions that affect epitope accessibility . Researchers should maintain detailed records of all experimental parameters to identify variables contributing to inconsistency, and consider validating results with alternative detection methods or antibodies targeting different epitopes of the same protein.

How can the At3g13820 antibody be used to study protein-protein interactions in Arabidopsis?

The At3g13820 antibody can be leveraged for multiple approaches to study protein-protein interactions:

• Co-immunoprecipitation (Co-IP):

  • Use the antibody to precipitate At3g13820 protein complexes

  • Identify interaction partners through mass spectrometry

  • Confirm specific interactions with Western blotting

• Proximity Ligation Assay (PLA):

  • Combine At3g13820 antibody with antibodies against potential interactors

  • Visualize protein interactions in situ with fluorescence microscopy

• Chromatin Immunoprecipitation (ChIP):

  • If At3g13820 functions in transcriptional complexes, use ChIP to identify associated DNA regions

• Immunofluorescence co-localization:

  • Use the antibody alongside markers for cellular compartments

  • Identify potential interaction sites through spatial co-localization

When designing such experiments, researchers should consider that F-box proteins like At3g13820 typically function in SCF (Skp1-Cullin-F-box) E3 ubiquitin ligase complexes that target proteins for degradation. Therefore, interactions may be transient and potentially stabilized by proteasome inhibitors. Additionally, researchers should validate observed interactions through multiple independent techniques, as each method has specific limitations and potential artifacts.

What factors might affect epitope accessibility when using the At3g13820 antibody in different applications?

Several factors can significantly impact epitope accessibility and antibody performance across different experimental applications:

• Fixation methods:

  • Chemical fixatives (formaldehyde, glutaraldehyde) may mask epitopes

  • Cross-linking can alter protein conformation

  • Duration and concentration of fixation affect epitope preservation

• Protein conformation:

  • Native vs. denatured states expose different epitopes

  • Post-translational modifications may block antibody binding sites

  • Protein-protein interactions might conceal recognition sites

• Sample preparation techniques:

  • Heat-induced epitope retrieval may be necessary for fixed tissues

  • Detergent types and concentrations affect membrane protein accessibility

  • pH conditions during processing can alter epitope structure

• Cellular localization:

  • Nuclear, cytoplasmic, or membrane localization requires different permeabilization strategies

  • Organelle-specific proteins may require specialized extraction methods

For flow cytometry applications, researchers should optimize cell preparation protocols considering that surface proteins and intracellular proteins require different permeabilization approaches . The validation strategy should confirm that the antibody can detect the target protein under the specific conditions of the intended application. When transitioning between applications (e.g., from Western blot to immunofluorescence), researchers should re-validate the antibody's performance as conditions affecting epitope accessibility differ substantially between techniques.

How can researchers optimize the At3g13820 antibody concentration for different applications?

Determining the optimal concentration of the At3g13820 antibody requires systematic titration for each application:

For each application, researchers should test multiple antibody concentrations while keeping all other variables constant. The optimal concentration provides the highest specific signal with minimal background. As noted in flow cytometry validation approaches, comparing the antibody's performance across cell lines with different target expression levels can help establish both sensitivity and specificity at various concentrations . Researchers should document the performance at each concentration and consider that different lots of the same antibody may require re-optimization. Additionally, the inclusion of appropriate positive and negative controls in titration experiments is essential for accurate determination of optimal working concentrations.

What approaches can be used to measure At3g13820 protein expression levels in Arabidopsis tissues?

Researchers can employ multiple complementary approaches to quantify At3g13820 protein expression:

• Western blot quantification:

  • Semi-quantitative analysis using the At3g13820 antibody

  • Normalization to housekeeping proteins (e.g., actin, tubulin)

  • Inclusion of recombinant protein standards for absolute quantification

• Flow cytometry:

  • Single-cell analysis of protein expression in protoplasts

  • Multiparametric analysis with cell type-specific markers

  • Comparative analysis across tissues or treatments

• ELISA-based quantification:

  • Development of sandwich ELISA using At3g13820 antibody

  • High-throughput analysis of multiple samples

  • Standard curve generation using recombinant protein

• Mass spectrometry:

  • Label-free quantification of digested peptides

  • Targeted approaches like selected reaction monitoring (SRM)

  • Comparison with proteomic databases

When validating antibody-based quantification methods, researchers should compare protein expression data with transcript levels while recognizing that post-transcriptional regulation may lead to discrepancies . For flow cytometry, cell tracker dyes can facilitate the comparison of different cell populations in mixed samples . Each quantification method has specific strengths and limitations, so researchers should select approaches based on required sensitivity, throughput, and whether relative or absolute quantification is needed.

How do different fixation and permeabilization protocols affect At3g13820 antibody performance?

Fixation and permeabilization protocols significantly impact antibody performance through multiple mechanisms:

For flow cytometry with the At3g13820 antibody, researchers must carefully optimize permeabilization to access intracellular targets while preserving epitope recognition . Different targets require tailored approaches - membrane proteins may be sensitive to harsh detergents, while nuclear proteins like transcription factors often require stronger permeabilization. Researchers should systematically test multiple fixation and permeabilization combinations to determine optimal conditions for their specific experimental system, starting with established protocols for plant proteins and refining based on empirical results.

What strategies can address potential cross-reactivity of the At3g13820 antibody?

To manage potential cross-reactivity of the At3g13820 antibody, researchers can implement several strategies:

• Pre-absorption controls:

  • Incubate antibody with purified antigen before use

  • Compare staining patterns with and without pre-absorption

  • Specific signals should disappear after pre-absorption

• Peptide competition assays:

  • Block antibody binding with excess immunizing peptide

  • Establish concentration-dependent blocking

  • Identify non-specific signals that persist despite blocking

• Cross-species validation:

  • Test antibody in species lacking the target or homologs

  • Identify non-specific binding patterns

• Multiple antibody validation:

  • Compare staining patterns with antibodies targeting different epitopes

  • Consistent patterns across antibodies suggest specificity

• Bioinformatic analysis:

  • Identify proteins with sequence similarity to immunizing peptide

  • Test antibody against recombinant versions of potential cross-reactive proteins

As antibody validation literature indicates, relying on multiple independent approaches provides the strongest evidence for specificity . For flow cytometry applications, comparing staining patterns in cell types with varying expression levels of the target protein can help distinguish specific from non-specific signals. Additionally, researchers should consider testing the antibody on tissues from knockout/knockdown lines of At3g13820 to confirm signal elimination, while being aware that incomplete knockdown may complicate interpretation .

How can researchers use the At3g13820 antibody to study protein dynamics during plant development?

The At3g13820 antibody can be used to examine protein dynamics across developmental stages through several approaches:

• Temporal expression profiling:

  • Sample collection at defined developmental stages

  • Quantitative Western blotting to track protein levels

  • Immunohistochemistry to observe spatial distribution changes

• Live cell imaging:

  • Antibody fragments or nanobodies for non-perturbing labeling

  • Pulse-chase experiments to track protein turnover

  • Correlation with developmental markers

• Proteasome-dependent degradation analysis:

  • Combine antibody detection with proteasome inhibitors

  • Measure protein half-life across developmental transitions

  • Identify regulation of F-box protein abundance itself

• Tissue-specific expression mapping:

  • Immunohistochemistry across tissue sections

  • Flow cytometry of protoplasts from different tissues

  • Co-staining with cell-type specific markers

For studying F-box proteins like At3g13820, which potentially function in protein degradation pathways, researchers should consider that the protein might exhibit dynamic regulation in response to developmental cues or environmental signals. Similar to nanobodies used in other research contexts, specialized antibody formats might allow for finer temporal resolution of protein dynamics . The experimental design should account for potentially transient expression patterns and include appropriate normalization to distinguish between actual changes in protein abundance versus variation in extraction efficiency between developmental stages.

What considerations are important when using the At3g13820 antibody for co-localization studies?

When performing co-localization studies with the At3g13820 antibody, researchers should address several critical factors:

• Antibody compatibility:

  • Select secondary antibodies with minimal cross-reactivity

  • Choose fluorophores with minimal spectral overlap

  • Consider sequential rather than simultaneous staining for problematic combinations

• Microscopy parameters:

  • Optimize image acquisition settings to minimize bleed-through

  • Use appropriate controls for spectral unmixing

  • Employ super-resolution techniques for closely associated proteins

• Quantitative co-localization analysis:

  • Calculate Pearson's correlation coefficients

  • Perform Manders' overlap coefficient analysis

  • Use randomization controls to establish significance thresholds

• Biological interpretation:

  • Distinguish between functional interaction and spatial proximity

  • Consider the resolution limits of optical microscopy

  • Validate with complementary biochemical approaches

F-box proteins like At3g13820 may localize to specific subcellular compartments, potentially including the nucleus, cytoplasm, or specific organelles depending on their function. When examining potential interactions with substrate proteins, researchers should consider that these interactions may be transient and potentially stabilized with proteasome inhibitors. For rigorous co-localization analysis, positive controls (known interacting proteins) and negative controls (proteins known not to interact) should be included to establish threshold values for meaningful co-localization.

How can researchers interpret conflicting results between At3g13820 antibody detection and transcript data?

Discrepancies between protein detection using At3g13820 antibody and corresponding mRNA expression may arise from several biological and technical factors:

• Post-transcriptional regulation mechanisms:

  • miRNA-mediated transcript degradation

  • Translational efficiency differences

  • Protein stability and turnover rates

• Temporal disconnects:

  • Delayed protein synthesis following transcription

  • Protein persistence after transcript degradation

  • Different sampling timepoints for RNA vs. protein

• Technical considerations:

  • Different detection sensitivities between methods

  • RNA extraction efficiency vs. protein extraction efficiency

  • Antibody specificity or accessibility issues

• Biological compartmentalization:

  • Spatial separation of transcription and translation

  • Protein trafficking affecting detection

  • Cell type heterogeneity in complex tissues

These discrepancies are well-documented in antibody validation literature, where RNAi knockdown efficiency at the RNA level does not always correspond to equivalent protein reduction . When investigating such conflicts, researchers should examine protein expression at multiple timepoints following observed transcript changes, as research on neutralizing antibodies has shown significant temporal dynamics in protein expression that may not align with transcript levels . Additionally, complementary approaches such as ribosome profiling or metabolic labeling can help bridge the gap between transcriptomic and proteomic data to provide mechanistic explanations for observed discrepancies.

What are the advanced applications of the At3g13820 antibody in plant stress response studies?

The At3g13820 antibody can be applied to several sophisticated approaches for studying plant stress responses:

• Stress-induced protein modifications:

  • Western blotting with phospho-specific or ubiquitin antibodies

  • Immunoprecipitation followed by mass spectrometry

  • Analysis of protein complex formation under stress

• Single-cell protein dynamics:

  • Flow cytometry of protoplasts from stressed tissues

  • Cell type-specific responses to stressors

  • Correlation with stress-induced transcription factors

• Spatial redistribution analysis:

  • Track subcellular localization changes during stress

  • Co-localization with stress granules or processing bodies

  • Nuclear-cytoplasmic shuttling quantification

• Temporal regulation studies:

  • Time-course analysis following stress application

  • Recovery phase protein dynamics

  • Correlation with physiological stress responses

As an F-box protein, At3g13820 may participate in the targeted degradation of regulatory proteins during stress responses. Researchers can adapt approaches used for studying antibody responses in disease models, where temporal dynamics critically influence outcomes . For instance, examining whether At3g13820 protein levels change during stress could indicate its role in stress adaptation. The experimental design should include appropriate controls for stress application, timing of sample collection, and quantification methods to detect potentially subtle or transient changes in protein abundance or localization in response to different stressors.