At3g16820 Antibody

What is the At3g16820 protein and why is it significant for plant research?

At3g16820 is a putative F-box protein encoded by the Arabidopsis thaliana genome (Uniprot ID: Q9LRZ2). Its significance stems from its involvement in ubiquitination pathways through its F-box domain, which typically mediates protein degradation via the SCF (Skp1, Cullin, F-box) complex. This protein plays a crucial role in plant development and stress responses by regulating protein turnover. Studying At3g16820 provides insights into plant cellular mechanisms that control growth, development, and adaptive responses.

What are the key specifications of commercially available At3g16820 antibodies?

The typical At3g16820 Antibody is a rabbit-derived polyclonal antibody with specific reactivity to Arabidopsis thaliana. It is validated for multiple applications including ELISA and Western Blot (WB). The antibody is commonly supplied in liquid form, purified using antigen affinity methods, and formulated in a storage buffer containing 50% glycerol, 0.01M PBS, and 0.03% Proclin 300 as a preservative. These specifications ensure stability and functionality in various experimental conditions while maintaining specificity for the target protein.

How is the At3g16820 antibody produced and what expression systems are used for the immunogen?

The At3g16820 antibody is produced by immunizing rabbits with a recombinant At3g16820 protein. The immunogen (antigen) is typically produced using multiple expression systems including yeast, E. coli, and mammalian cells to ensure proper protein folding and epitope presentation. The full-length At3g16820 protein sequence is used as the immunogen, which enables the resulting antibody to recognize multiple epitopes in both native and denatured forms of the protein. This production method yields antibodies with high specificity and affinity for various experimental applications.

What is the optimal protocol for Western blot analysis using At3g16820 antibody?

For optimal Western blot results with At3g16820 antibody, follow this methodological approach:

• Sample Preparation: Extract total protein from Arabidopsis tissues using a buffer containing protease inhibitors to prevent degradation

• Protein Separation: Load 20-50 μg protein per lane on SDS-PAGE (10-12%)

• Transfer: Use PVDF membrane (0.45 μm) with semi-dry transfer at 15V for 30 minutes

• Blocking: Block with 5% non-fat milk in TBST for 1 hour at room temperature

• Primary Antibody: Dilute At3g16820 antibody 1:50 to 1:500 in blocking solution; incubate overnight at 4°C

• Washing: Wash membrane 3-5 times with TBST, 5 minutes each

• Secondary Antibody: Incubate with HRP-conjugated anti-rabbit IgG (1:5000) for 1 hour at room temperature

• Detection: Use ECL substrate and expose to X-ray film or digital imager

This protocol accounts for the potentially high working dilution (1:50) that may be required for optimal signal detection with this polyclonal antibody.

How should the At3g16820 antibody be validated for specificity in plant research?

A comprehensive validation strategy for At3g16820 antibody should include:

• Positive Control: Use purified recombinant At3g16820 protein to confirm antibody binding

• Negative Control: Test antibody against protein extracts from At3g16820 knockout plants

• Peptide Competition Assay: Pre-incubate antibody with immunizing peptide/protein to block specific binding

• Cross-Reactivity Assessment: Test against protein extracts from other plant species to confirm specificity to Arabidopsis

• Multiple Detection Methods: Validate using different techniques (Western blot, ELISA, immunoprecipitation)

• Comparison with Epitope-Tagged Protein: Express At3g16820 with an epitope tag and compare detection patterns

These validation steps ensure experimental results are reliable and specific to the target protein, preventing misinterpretation of data in plant research applications.

What are the recommended ELISA protocols for quantifying At3g16820 protein levels?

For quantitative analysis of At3g16820 protein using ELISA:

• Plate Coating: Coat high-binding ELISA plates with capture antibody (2-5 μg/ml) in carbonate buffer (pH 9.6) overnight at 4°C

• Blocking: Block with 2-3% BSA in PBS for 1-2 hours at room temperature

• Sample Addition: Add protein extracts and standards in dilution buffer; incubate 2 hours at room temperature

• Detection Antibody: Apply At3g16820 antibody at 1:500 dilution; incubate 2 hours at room temperature

• Secondary Antibody: Add HRP-conjugated anti-rabbit IgG; incubate 1 hour

• Substrate Addition: Add TMB substrate and monitor color development

• Signal Reading: Stop reaction with H₂SO₄ and read absorbance at 450nm

To ensure reliable results, include proper controls and avoid sodium azide in buffers as it inhibits HRP activity . Thorough washing between steps is critical to reduce background and increase signal-to-noise ratio.

How can researchers address weak or no signal issues when using At3g16820 antibody in ELISA or Western blot?

When encountering weak or absent signals with At3g16820 antibody, implement these methodological solutions:

• Antibody Concentration: Increase antibody concentration starting with 1:50 dilution for Western blots; concentrate antibody using commercially available concentration kits if necessary

• Incubation Conditions: Extend primary antibody incubation to overnight at 4°C to enhance binding

• Antigen Retrieval: For fixed samples, optimize antigen retrieval methods to expose epitopes

• Protein Loading: Increase protein concentration in samples to enhance detection sensitivity

• Detection System: Use more sensitive detection reagents (e.g., switch from colorimetric to chemiluminescent)

• Sample Preparation: Ensure protein extraction method preserves the integrity of At3g16820 protein

• Antibody Storage: Verify antibody hasn't been repeatedly freeze-thawed, which can reduce epitope-binding capacity

If signal remains weak, perform a titration series of both primary and secondary antibodies to determine optimal working concentrations for your specific experimental conditions .

What strategies can minimize high background when using At3g16820 antibody?

To effectively reduce high background with At3g16820 antibody:

How should At3g16820 antibody be properly stored and handled to maintain activity?

For optimal preservation of At3g16820 antibody activity:

• Storage Temperature: Maintain at -20°C or -80°C long-term in the provided 50% glycerol-PBS buffer to prevent aggregation

• Aliquoting: Upon receipt, divide into small working aliquots to avoid repeated freeze-thaw cycles

• Freeze-Thaw Cycles: Minimize freeze-thaw events as they significantly reduce epitope-binding capacity

• Working Dilutions: Prepare fresh working dilutions on the day of experiment

• Buffer Composition: Verify storage buffer contains proper preservatives (e.g., 0.03% Proclin 300)

• Shelf Life: Monitor expiration dates; typical shelf life is 12 months under recommended conditions

• Temperature Fluctuations: Avoid prolonged exposure to room temperature during experiments

Additionally, centrifuge antibody vials briefly before opening to collect liquid at the bottom of the tube, especially after shipping or long storage periods.

How can At3g16820 antibody be utilized for studying plant ubiquitination pathways?

The At3g16820 antibody can be strategically employed to investigate plant ubiquitination pathways through:

• Co-immunoprecipitation (Co-IP): Pull down At3g16820 protein complexes to identify interaction partners within the SCF complex, revealing the substrate specificity of this F-box protein

• Chromatin Immunoprecipitation (ChIP): If At3g16820 has nuclear localization, determine if it associates with chromatin to regulate gene expression

• Immunohistochemistry (IHC): Visualize tissue-specific expression patterns of At3g16820 during plant development and stress responses

• Protein Stability Assays: Monitor At3g16820 protein levels under various conditions to determine factors affecting its turnover

• Proteasome Inhibition Studies: Compare At3g16820 levels with and without proteasome inhibitors to confirm its regulation through the ubiquitin-proteasome system

• Pulse-Chase Experiments: Track protein degradation rates of potential At3g16820 substrates using radioisotope labeling

These advanced applications leverage the specificity of the At3g16820 antibody to dissect complex molecular pathways in plant cellular regulation, particularly those involving targeted protein degradation.

What considerations should be made when using At3g16820 antibody for comparative studies across different plant species?

When extending At3g16820 antibody research to comparative plant studies:

• Sequence Homology Analysis: Perform bioinformatic analysis of At3g16820 homologs across target species to predict cross-reactivity potential

• Epitope Conservation: Evaluate conservation of specific epitopes recognized by the antibody in homologous proteins

• Validation in Each Species: Rigorously validate antibody specificity in each new species using knockout/knockdown lines when available

• Western Blot Optimization: Adjust protein extraction protocols for different plant tissues, accounting for varying metabolite compositions

• Signal Normalization: Implement consistent loading controls appropriate for cross-species comparisons

• Negative Controls: Include samples from phylogenetically distant species as negative controls

• Sensitivity Calibration: Determine detection limits in each species by using purified recombinant proteins

While the At3g16820 antibody is specifically validated for Arabidopsis thaliana, these methodological approaches can help assess and potentially extend its utility to comparative studies with appropriate controls and validations.

How can researchers combine At3g16820 antibody with advanced imaging techniques for subcellular localization studies?

To leverage At3g16820 antibody for high-resolution subcellular localization:

• Immunogold Electron Microscopy:

  • Fix plant tissues in 4% paraformaldehyde/0.1% glutaraldehyde

  • Embed in LR White resin and prepare ultrathin sections

  • Incubate with At3g16820 antibody (1:20-1:50 dilution)

  • Apply gold-conjugated secondary antibody

  • This approach provides nanometer-scale resolution of protein localization

• Super-Resolution Microscopy:

  • Use fluorophore-conjugated secondary antibodies compatible with STORM or PALM

  • Implement sample clearing techniques to enhance signal-to-noise ratio

  • Apply drift correction during image acquisition

  • These techniques can resolve protein clusters below the diffraction limit

• Multiplex Immunofluorescence:

  • Combine At3g16820 antibody with antibodies against organelle markers

  • Use spectral unmixing to separate overlapping fluorescence signals

  • Apply deconvolution algorithms to enhance resolution

  • This approach contextualizes At3g16820 localization within cellular compartments

While not directly tested for axon identification, analogous antibody applications demonstrate potential for cellular marker studies in model organisms, which could be extended to plant cell biology research with appropriate modifications.

How should researchers interpret variations in At3g16820 protein expression patterns between different plant tissues?

When analyzing differential At3g16820 expression across plant tissues:

• Normalization Strategy: Implement multiple housekeeping proteins as loading controls that maintain stability across different tissue types

• Tissue-Specific Extraction Optimization: Adjust extraction protocols to account for varying tissue compositions (e.g., higher polysaccharide content in fruits vs. leaves)

• Developmental Context: Interpret expression patterns within the developmental stage and physiological state of each tissue

• Statistical Analysis: Apply appropriate statistical tests (ANOVA with post-hoc tests) to determine significant differences between tissues

• Biological Replicates: Analyze at least three independent biological replicates to account for natural variation

• Comparative Quantification: Use densitometry for Western blots or fluorescence intensity measurements for immunohistochemistry to quantify relative expression levels

Variations in At3g16820 protein levels may reflect tissue-specific roles in protein degradation pathways, developmental regulation, or responses to environmental conditions. Correlate protein expression patterns with known developmental or stress-responsive gene expression datasets to provide contextual interpretation.

What control experiments are essential when studying protein-protein interactions involving At3g16820?

For robust protein-protein interaction studies involving At3g16820:

• Input Controls: Analyze 5-10% of pre-immunoprecipitation lysate to confirm target protein presence

• Negative Controls:

  • IgG control: Use non-specific IgG from the same species as At3g16820 antibody

  • Knockout/knockdown controls: Use plant materials lacking At3g16820 expression

  • Peptide competition: Pre-incubate antibody with immunizing peptide

• Reciprocal Co-IP: Confirm interactions by immunoprecipitating with antibodies against the putative interacting partner

• Denaturing Controls: Perform interactions under denaturing conditions to identify direct vs. indirect interactions

• Crosslinking Validation: Compare results with and without protein crosslinking to capture transient interactions

• Domain Mutants: Use truncated versions of At3g16820 to map interaction domains

These methodological controls help distinguish specific interactions from experimental artifacts, ensuring that identified protein-protein interactions involving At3g16820 are biologically relevant and not technical artifacts.