At2g35280 Antibody
Description
Overview of At2g35280 Antibody
The At2g35280 Antibody is a monoclonal antibody specifically targeting the F-box protein At2g35280 (Q1PEW8) in Arabidopsis thaliana. This antibody is designed for research applications, particularly in Western blotting (WB) and immunoprecipitation (IP), to study protein interactions and degradation pathways mediated by the ubiquitin-proteasome system.
Key Parameters
Application Details
| Application | Recommendations | Performance |
|---|---|---|
| Western Blot | Starting dilution: 1:1000; Detects 0.01–1 ng of immunogen peptide in dot blot | ELISA titer: 10,000 |
| Immunoprecipitation | Covered under AbInsure™ program (requires validation) | Not explicitly tested in provided data |
Availability and Pricing
| Aspect | Details | Source |
|---|---|---|
| Package Name | X1-Q1PEW8 [ABX] (0.2 mg) | |
| Price | $899 (X2 package) + $100 shipping | |
| Lead Time | 14–16 weeks (made-to-order) |
Research Applications and Context
The At2g35280 Antibody is tailored for studying F-box proteins, which are critical components of the SCF (Skp1-Cullin-F-box) E3 ubiquitin ligase complexes. These complexes regulate protein turnover in plants, influencing processes such as stress responses and developmental transitions. While specific studies using this antibody are not detailed in the provided sources, its utility lies in:
-
Protein Localization: Identifying At2g35280 in subcellular compartments.
-
Interactome Analysis: Mapping interactions with other E3 ligase components (e.g., Skp1, Cullin).
-
Phytohormone Signaling: Investigating roles in hormone-regulated protein degradation.
Validation and Specificity Considerations
Commercial antibodies often face challenges in specificity, as seen in studies of angiotensin II AT2 receptor antibodies . For At2g35280 Antibody:
-
Specificity: Targets the N terminus of Q1PEW8, minimizing cross-reactivity with related F-box proteins.
-
Quality Assurance: Part of the AbInsure™ program, which guarantees performance in WB and IP (post-validation).
Comparative Analysis with Other F-Box Antibodies
| Feature | At2g35280 Antibody | Generic F-Box Antibodies |
|---|---|---|
| Target Specificity | N-terminal At2g35280 (Q1PEW8) | Broad F-box motifs (cross-reactive) |
| Application Range | WB/IP (validated for At2g35280) | WB, IHC (variable performance) |
| Customization | Synthetic peptide immunogens (sequence-defined) | Polyclonal or pan-F-box approaches |
Product Specs
Constituents: 50% Glycerol, 0.01M Phosphate Buffered Saline (PBS), pH 7.4
Q&A
Here’s a structured FAQ collection for researchers working with the At2g35280 antibody in plant biology, synthesized from current antibody research principles and tailored to academic scenarios. While no direct studies on At2g35280 antibodies were found, the answers draw parallels from monoclonal antibody development and validation methodologies in the provided sources.
How to validate the specificity of an At2g35280 antibody in Arabidopsis thaliana?
Methodology:
-
Perform immunoblotting using protein extracts from wild-type and At2g35280 knockout mutants. A specific antibody will show a band in wild-type but not in mutants .
-
Use peptide competition assays: Pre-incubate the antibody with the immunizing peptide to confirm signal loss.
-
Validate cross-reactivity by testing against homologous F-box proteins (e.g., At1g27380, At3g61060) .
Key Parameters:
| Validation Step | Expected Outcome | Tool/Reagent |
|---|---|---|
| Western Blot | Single band at predicted molecular weight (~50 kDa) | At2g35280 knockout line |
| Immunofluorescence | Subcellular localization consistent with F-box protein function (e.g., nucleus/cytoplasm) | Confocal microscopy |
What experimental designs are optimal for studying At2g35280 protein-protein interactions?
Approach:
-
Co-Immunoprecipitation (Co-IP): Use seedlings treated with proteasome inhibitors (e.g., MG132) to stabilize transient interactions .
-
Bimolecular Fluorescence Complementation (BiFC): Co-express At2g35280-YN and candidate partner-YC in protoplasts to map interaction sites .
-
Include negative controls: (i) Empty vector transfections, (ii) Non-interacting F-box proteins.
Common Pitfalls:
-
False positives due to antibody cross-reactivity (address via knockout validation).
-
Overexpression artifacts (use endogenous promoters for transgenic lines).
How to resolve contradictory data in At2g35280 functional studies?
Case Example: Antibody detects protein in roots but not leaves.
Troubleshooting:
-
Tissue-specific degradation: Test protein stability using cycloheximide chase assays .
-
Epitope masking: Compare antibody performance in native vs. denaturing conditions (e.g., formaldehyde-fixed vs. frozen sections) .
-
Alternative splicing: Check for isoform-specific antibodies via RNA-seq alignment .
Data Reconciliation Framework:
| Observation | Hypothesis | Test |
|---|---|---|
| Signal variability across tissues | Tissue-specific post-translational modifications | Phos-tag SDS-PAGE |
| Discrepant localization reports | Antibody cross-reactivity | CRISPR-Cas9 fluorescent tagging |
Can computational modeling guide At2g35280 antibody engineering for improved affinity?
Strategy:
-
Use RosettaAntibody or OptCDR to redesign complementarity-determining regions (CDRs) targeting conserved F-box motifs .
-
Validate predictions via surface plasmon resonance (SPR) to measure binding kinetics (KD ≤ 10 nM target) .
-
Prioritize mutations that enhance electrostatic complementarity (e.g., arginine substitutions near acidic epitopes) .
Design Workflow:
-
Generate homology model of At2g35280 using AlphaFold2.
-
Identify solvent-exposed epitopes with >90% sequence conservation.
-
Simulate CDR-epitope interactions and select top 5 designs for synthesis.
How to adapt At2g35280 antibodies for multiplexed imaging in live cells?
Innovative Application:
-
Engineer nanobodies (single-domain antibodies) fused to fluorescent proteins (e.g., GFP, mScarlet) for real-time tracking .
-
Use pH-sensitive dyes (e.g., pHluorin) to monitor At2g35280 trafficking through endosomal compartments.
-
Validate with fluorescence correlation spectroscopy (FCS) to quantify binding dynamics .
Optimization Table:
| Parameter | Requirement | Solution |
|---|---|---|
| Brightness | ≥50,000 photons/ms | Tandem dimer nanobody design |
| Photostability | >5 min continuous imaging | mEos4a fusion |
| Specificity | Zero cross-talk with SKP1/ASK1 | Epitope grafting from |
Methodological Notes
-
Antibody Validation: Always pair antibody-based data with genetic complementation assays (e.g., At2g35280 rescue lines) .
-
Data Reproducibility: Archive aliquots at −80°C with 0.02% sodium azide to prevent aggregation .
-
Ethical Reporting: Disclose antibody lot numbers and validation data in supplementary materials.
