At3g24700 Antibody

What is At3g24700 and why is it significant for plant research?

At3g24700 is a gene that encodes a putative F-box protein in Arabidopsis thaliana (Mouse-ear cress), a model organism extensively used in plant molecular biology. The protein is part of the F-box protein family, which typically functions within SCF (Skp1-Cullin-F-box) complexes that mediate protein ubiquitination and subsequent degradation through the 26S proteasome pathway. F-box proteins play crucial roles in multiple cellular processes including cell cycle regulation, hormone signaling, and development in plants. Research on At3g24700 contributes to our understanding of protein degradation pathways and their regulatory roles in plant biology .

Methodologically, studying At3g24700 requires specific antibodies that can reliably detect the protein in various experimental contexts. The commercially available At3g24700 antibody (such as CSB-PA873278XA01DOA) is generated against recombinant Arabidopsis thaliana At3g24700 protein and purified through antigen affinity methods .

What are the validated applications for At3g24700 antibody?

The At3g24700 antibody has been validated for several experimental applications, with the primary validated methods being:

• Enzyme-Linked Immunosorbent Assay (ELISA) - For quantitative detection of At3g24700 protein in solution

• Western Blot (WB) - For detection of denatured At3g24700 protein separated by electrophoresis

When designing experiments, researchers should consider that antibody validation typically follows a multi-pillar approach as recommended by experts in the field. This involves confirming specificity through:

• Genetic strategies (knockouts/knockdowns)

• Orthogonal targeting methods

• Independent antibody verification

• Expression of tagged proteins

• Immunocapture mass spectrometry

For At3g24700 specifically, validation data should be reviewed prior to experimental design, as comprehensive validation across all possible applications may not be available.

How should I optimize Western blot protocols for At3g24700 detection?

Optimizing Western blot protocols for At3g24700 detection requires careful consideration of multiple parameters:

Sample Preparation:

• Extract proteins from Arabidopsis tissues using an appropriate buffer containing protease inhibitors to prevent degradation

• Determine optimal protein concentration (typically 20-50 μg total protein)

• Denature samples completely in Laemmli buffer with β-mercaptoethanol at 95°C for 5 minutes

Electrophoresis and Transfer:

• Use an appropriate percentage gel based on At3g24700's predicted molecular weight

• Include positive and negative controls (when available)

• Transfer to PVDF or nitrocellulose membrane using standard protocols

Antibody Incubation:

• Block membrane with 5% non-fat dry milk or BSA in TBST for 1 hour at room temperature

• Incubate with At3g24700 antibody at recommended dilution (determine empirically, starting with manufacturer recommendations)

• Incubate overnight at 4°C with gentle rocking

• Wash thoroughly with TBST (at least 3 × 10 minutes)

• Incubate with appropriate secondary antibody (anti-rabbit IgG for the polyclonal antibody described in the search results)

Detection and Troubleshooting:

• Use enhanced chemiluminescence (ECL) or fluorescent detection methods

• If background is high, increase blocking time or washing steps

• If signal is weak, optimize antibody concentration or protein loading

When optimizing, remember that the At3g24700 antibody is preserved in a buffer containing 50% glycerol and 0.03% Proclin 300 in 0.01M PBS at pH 7.4 . These components may influence antibody performance and should be considered when troubleshooting.

What experimental controls are essential when using At3g24700 antibody for immunological detection?

Implementing proper controls is essential for reliable interpretation of results when using At3g24700 antibody:

Positive Controls:

• Recombinant At3g24700 protein (when available)

• Lysates from tissues/cells known to express At3g24700

• Overexpression systems (e.g., Arabidopsis plants or cell lines transfected with At3g24700 expression constructs)

Negative Controls:

• Tissues from At3g24700 knockout/knockdown plants

• Pre-immune serum controls

• Secondary antibody-only controls

• Blocking peptide competition assays

Validation Controls:

• Using orthogonal methods of detection (e.g., RNA expression correlation with protein levels)

• Comparison with different antibodies against the same target

• Immunoprecipitation followed by mass spectrometry to confirm identity

For experimental validation, employing multiple approaches as outlined in antibody validation frameworks is recommended. The "five pillars" approach suggests using genetic, orthogonal, independent antibody, tagged protein expression, and immunocapture methods to establish confidence in antibody specificity .

A methodological workflow for proper controls might include:

• Initial screening in genetically modified cell types

• Verification that observed staining represents the target in the sample of interest

• Correlation of antibody signals with expected expression patterns across tissues or experimental conditions

How do I validate At3g24700 antibody specificity for my specific experimental system?

Validating antibody specificity for At3g24700 in your specific experimental system requires a multi-faceted approach:

Genetic Validation:

• Generate or obtain At3g24700 knockout/knockdown lines

• Compare antibody signal between wild-type and knockout/knockdown samples

• Observe reduction or elimination of signal in genetic knockout/knockdown systems

Expression System Validation:

• Create overexpression constructs for At3g24700

• Transiently or stably express the construct in your experimental system

• Verify increased antibody signal corresponding to overexpression

Cross-Reactivity Assessment:

• Perform bioinformatic analysis to identify related proteins that might cross-react

• Test the antibody against recombinant related proteins

• Evaluate specificity across multiple species if working in comparative systems

Application-Specific Validation:
For flow cytometry applications, consider approaches like those recommended by experts:

• Compare antibody labeling across cell lines expressing different levels of the target

• Use cell tracker dyes to mix cell populations with different expression levels

• Correlate antibody labeling with orthogonal data (e.g., RNA-seq expression data)

For Western blot applications, additional validation might include:

• Testing antibody performance across a range of protein concentrations

• Verifying size specificity by comparing observed band size with predicted molecular weight

• Performing peptide competition assays to confirm epitope specificity

What are the advanced techniques for analyzing At3g24700 protein interactions and function?

Investigating At3g24700 protein interactions and functions requires sophisticated techniques beyond basic detection:

Protein-Protein Interaction Analysis:

• Co-immunoprecipitation (Co-IP) using At3g24700 antibody to pull down interaction partners

• Yeast two-hybrid screening to identify potential interactors

• Proximity-based labeling methods (BioID, APEX) to identify proximal proteins in vivo

• Förster Resonance Energy Transfer (FRET) or Bimolecular Fluorescence Complementation (BiFC) for monitoring interactions in living cells

Functional Analysis:

• Chromatin Immunoprecipitation (ChIP) if At3g24700 is involved in transcriptional regulation

• Immunoprecipitation coupled with mass spectrometry (IP-MS) to identify post-translational modifications

• Protein stability assays to investigate potential roles in protein degradation pathways

Subcellular Localization:

• Immunofluorescence microscopy using the At3g24700 antibody

• Co-localization studies with known subcellular markers

• Live-cell imaging with fluorescently tagged At3g24700 constructs

Since At3g24700 is putatively identified as an F-box protein, techniques specifically designed to investigate protein degradation pathways would be particularly relevant. These might include ubiquitination assays, proteasome inhibition studies, and protein half-life measurements in various genetic backgrounds.

What are the critical storage and handling guidelines for maximizing At3g24700 antibody performance?

Proper storage and handling of At3g24700 antibody is crucial for maintaining its performance over time:

Storage Conditions:

• Store at -20°C or -80°C upon receipt

• Avoid repeated freeze-thaw cycles that can degrade antibody activity

• Consider preparing small working aliquots to minimize freeze-thaw cycles

Handling Guidelines:

• Always keep antibodies on ice when in use

• Return to appropriate storage temperature promptly after use

• Use sterile technique when handling antibody solutions

• Centrifuge briefly before opening vials to collect liquid at the bottom

Working Solution Preparation:

• Prepare fresh dilutions in appropriate buffer for each experiment

• Include stabilizing proteins (such as BSA) in working solutions

• Document lot number, dilution factors, and preparation dates

Long-term Considerations:

• Monitor antibody performance over time with consistent positive controls

• Maintain detailed records of antibody performance across experiments

• Validate new lots against previous lots before complete transition

The At3g24700 antibody is supplied in a liquid form with 50% glycerol, 0.03% Proclin 300, and 0.01M PBS at pH 7.4 . This formulation helps maintain stability, but proper storage remains essential.

How do I determine the optimal working dilution for At3g24700 antibody in different applications?

Determining optimal working dilution for At3g24700 antibody requires systematic titration across applications:

Western Blot Titration:

• Prepare a dilution series (e.g., 1:500, 1:1000, 1:2000, 1:5000)

• Use consistent protein samples across all dilutions

• Evaluate signal-to-noise ratio at each dilution

• Select the dilution that provides clear specific signal with minimal background

ELISA Titration:

• Create an antibody dilution series (wider range than for Western blot)

• Maintain consistent antigen concentration

• Plot antibody dilution versus signal strength

• Identify the dilution in the linear range of detection

Titration Experiment Design:

Complete this table with experimental data from your system to determine optimal dilutions.

Optimization Factors:

• Different applications typically require different dilutions

• Sample type may influence optimal dilution (e.g., recombinant protein vs. tissue extract)

• Detection method sensitivity impacts required antibody concentration

• Background levels in different sample types may necessitate adjustment

What are common issues encountered with At3g24700 antibody and their solutions?

Researchers working with At3g24700 antibody may encounter several challenges that require systematic troubleshooting:

High Background Issues:

• Insufficient blocking: Extend blocking time or increase blocking agent concentration

• Inadequate washing: Increase number and duration of wash steps

• Secondary antibody cross-reactivity: Test different secondary antibodies or increase dilution

• Non-specific binding: Add carrier proteins (BSA) to antibody dilution

Weak or No Signal:

• Protein degradation: Ensure proper sample handling and add protease inhibitors

• Insufficient protein: Increase loading amount or concentrate samples

• Inefficient transfer: Optimize transfer conditions for the protein's molecular weight

• Epitope masking: Try different sample preparation methods or denaturing conditions

• Antibody degradation: Test new antibody aliquot or lot

Multiple or Unexpected Bands:

• Protein degradation: Improve sample preparation with fresh protease inhibitors

• Post-translational modifications: Verify with literature or additional experiments

• Cross-reactivity: Perform additional validation with knockout/knockdown samples

• Splice variants: Compare with genomic data on potential isoforms

Inconsistent Results:

• Variable protein expression: Standardize growth/treatment conditions

• Inconsistent loading: Verify with loading controls

• Antibody degradation: Aliquot antibody and minimize freeze-thaw cycles

• Variable transfer efficiency: Use stained membranes to verify transfer

How can I adapt At3g24700 antibody protocols for different plant species or tissue types?

Adapting At3g24700 antibody protocols across species or tissues requires consideration of multiple factors:

Cross-Species Application:

• Perform sequence alignment of At3g24700 across target species

• Identify epitope conservation if epitope information is available

• Test antibody reactivity empirically with samples from each species

• Optimize extraction buffers for different species (considering cell wall composition differences)

Tissue-Specific Considerations:

• Modify extraction protocols based on tissue characteristics:

  • Leaf tissue: Standard extraction may be sufficient

  • Root tissue: May require additional steps to remove soil contaminants

  • Seed tissue: Often needs harsher extraction conditions due to dense storage proteins

  • Floral tissue: May contain higher levels of secondary metabolites requiring modification

• Adjust protein extraction buffers:

  • High-phenolic tissues: Add polyvinylpyrrolidone (PVP) or polyvinylpolypyrrolidone (PVPP)

  • Starch-rich tissues: Include additional centrifugation steps

  • Lipid-rich tissues: Consider additional detergent or organic extraction steps

Validation Across Systems:

• Confirm antibody specificity in each new system

• Use positive controls from Arabidopsis thaliana alongside new samples

• Consider generating system-specific positive controls (overexpression constructs)

• Document performance differences across systems for reproducibility

How should I interpret quantitative differences in At3g24700 protein levels across experimental conditions?

Interpreting quantitative differences in At3g24700 protein levels requires rigorous analytical approaches:

Quantification Methods:

• Densitometry analysis of Western blot bands

• Quantitative ELISA measurements

• Normalization to appropriate housekeeping proteins or total protein

• Statistical analysis across biological and technical replicates

Experimental Design Considerations:

• Include sufficient biological replicates (minimum n=3, preferably more)

• Perform technical replicates to assess method variability

• Include appropriate controls for normalization

• Design experiments to account for potential confounding variables

Interpretation Framework:

• Compare relative changes rather than absolute values

• Consider biological significance of fold-changes (statistical vs. biological significance)

• Correlate protein changes with phenotypic or physiological outcomes

• Integrate data with other molecular measurements (transcript levels, protein interactions)

Documentation and Reporting:
Create comprehensive data tables that include:

Complete this table with experimental data from your system for proper interpretation.

What are the cutting-edge applications of At3g24700 antibody in plant molecular biology research?

The At3g24700 antibody enables several advanced research applications in plant molecular biology:

Systems Biology Integration:

• Proteomics profiling following environmental stress or developmental transitions

• Integration of protein-level data with transcriptomics and metabolomics

• Network analysis of F-box protein involvement in regulatory pathways

• Comparison of protein dynamics across mutant backgrounds

Developmental Biology Applications:

• Tissue-specific and temporal expression pattern analysis

• Correlation of protein levels with developmental transitions

• Investigation of protein regulation during embryogenesis or flowering

• Analysis of protein redistribution during cellular differentiation

Stress Response Studies:

• Quantification of protein level changes under various abiotic stresses

• Investigation of post-translational modifications in response to stress

• Protein stability and turnover analysis under stress conditions

• Subcellular relocalization studies during stress response

Evolutionary Biology Applications:

• Comparative analysis of F-box protein conservation across plant species

• Investigation of functional conservation or divergence

• Study of protein adaptation across environmental gradients

• Analysis of protein evolution in cultivated versus wild species

The use of At3g24700 antibody in these applications should incorporate principles of antibody validation appropriate to each experimental context, following the frameworks established in the antibody research community .