At1g21990 Antibody
Description
Introduction to At1g21990 Antibody
The "At1g21990 Antibody" refers to an antibody targeting a specific protein encoded by the gene At1g21990 in Arabidopsis thaliana. This gene is involved in various plant physiological processes, but specific details about the antibody itself, such as its application, structure, or function, are not widely documented in the provided search results. Therefore, this article will focus on the general principles of antibodies and their applications, while also discussing the potential relevance of antibodies in plant biology.
General Principles of Antibodies
Antibodies, also known as immunoglobulins, are proteins produced by the immune system to recognize and bind to specific antigens. They are composed of four polypeptide chains: two heavy chains and two light chains, forming a Y-shaped structure with two antigen-binding sites . The variable regions of these chains determine the specificity of the antibody for its antigen.
| Antibody Class | Heavy Chain Class | Molecular Weight (kDa) | % Total Serum Antibody | Functional Properties |
|---|---|---|---|---|
| IgM | μ (mu) | 900 | 6 | Agglutination, Complement Activation |
| IgG | γ (gamma) | 150 | 80 | Neutralization, Opsonization, Complement Activation |
| IgA | α (alpha) | 385 | 13 | Mucosal Immunity |
| IgE | ε (epsilon) | 200 | 0.002 | Allergic Reactions |
| IgD | δ (delta) | 180 | 1 | Antigen Recognition on B Cells |
Applications of Antibodies
Antibodies are widely used in research, diagnostics, and therapy due to their specificity and versatility. They can be used to detect specific proteins, neutralize pathogens, or deliver drugs to targeted cells.
Research Applications
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Western Blotting: Antibodies are used to detect specific proteins in cell extracts.
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Immunohistochemistry: Antibodies help visualize proteins in tissue sections.
Therapeutic Applications
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Monoclonal Antibodies: Engineered to target specific antigens, these are used in treatments for cancer, autoimmune diseases, and infections.
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Antibody-Dependent Cellular Cytotoxicity (ADCC): Antibodies can direct immune cells to destroy target cells.
Potential Relevance of At1g21990 Antibody in Plant Biology
While specific information on the At1g21990 Antibody is limited, antibodies in general play a crucial role in plant biology research. They can be used to study plant proteins, understand plant-pathogen interactions, and develop diagnostic tools for plant diseases.
Plant Protein Studies
Antibodies can be used to study the expression and localization of plant proteins involved in stress responses, development, or metabolic pathways.
Plant-Pathogen Interactions
Antibodies can help identify proteins involved in plant defense mechanisms or pathogen virulence factors.
Product Specs
Q&A
How can researchers validate the specificity of At1g21990 antibodies in plant protein extracts?
To validate specificity, perform immunoblotting and immunoprecipitation alongside negative controls. For Camelina sativa studies, use wild-type and At1g21990 knockout lines to confirm antibody binding absence in knockouts . Include recombinant At1g21990 protein as a positive control. Cross-reactivity risks arise due to F-box protein family homology, so employ peptide competition assays: pre-incubate the antibody with excess antigenic peptide (e.g., residues 50-75 of At1g21990) to block binding. Quantify band intensity reduction ≥80% to confirm specificity .
Key Validation Parameters:
| Assay | Control Type | Acceptance Criterion |
|---|---|---|
| Western Blot | Knockout plant extract | No detectable bands |
| ELISA | Recombinant protein | Linear range: 0.1–10 µg/mL |
| Immunofluorescence | Non-transgenic tissue | Signal intensity ≤5% of test |
What experimental designs are optimal for assessing At1g21990 protein-protein interactions?
Use yeast two-hybrid screens with At1g21990 as bait against a cDNA library from target tissues. For in planta validation, apply bimolecular fluorescence complementation (BiFC) in Nicotiana benthamiana leaves. Critical considerations:
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pH optimization: F-box proteins like At1g21990 often interact with SKP1-like partners under pH 7.2–7.6 .
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Proteasome inhibition: Treat samples with 10 µM MG-132 for 4 hr pre-harvest to stabilize transient interactions .
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Quantitative co-IP: Pair with mass spectrometry (MS) for interaction stoichiometry analysis. A ≥2-fold enrichment of co-precipitated proteins vs. IgG controls indicates significance .
How should researchers address contradictory data on At1g21990 subcellular localization?
Contradictions often stem from fixation artifacts or epitope masking. Triangulate results using:
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Live-cell imaging: Fuse At1g21990 with GFP under its native promoter.
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Fractionation assays: Compare nuclear/cytoplasmic ratios via differential centrifugation (e.g., 1,000×g pellet for nuclei).
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Transient overexpression: Tag with dual markers (e.g., mCherry-NLS vs. GFP-At1g21990) to rule out mislocalization artifacts .
Localization Data Comparison Framework:
| Method | Observed Localization | Potential Bias |
|---|---|---|
| Immunofluorescence | Nucleus | Fixation-induced epitope exposure |
| GFP Fusion | Cytoplasm | Overexpression artifacts |
| Subcellular Fractionation | Both compartments | Cross-contamination risks |
What strategies resolve glycoform-related variability in At1g21990 antibody binding affinity?
At1g21990’s putative F-box domain contains N-glycosylation sites (e.g., Asn-102). To assess glycoform impacts:
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Glycoengineered models: Express At1g21990 in Arabidopsis glycosylation mutants (cgl1 for oligomannose; xylt/fuct for complex glycans) .
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Surface plasmon resonance (SPR): Compare antibody binding kinetics (KD) between glycoforms. For example, Man5 glycoforms show 2.6-fold faster off-rates vs. G0F variants .
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Glycan masking assays: Treat purified protein with PNGase F or endo H; ≥30% signal reduction in ELISA indicates glycan-dependent epitopes .
How can cryo-EM guide epitope mapping for At1g21990 antibodies?
For antibodies targeting conformational epitopes:
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Complex preparation: Incubate At1g21990 with Fab fragments at 3:1 molar excess.
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Grid optimization: Use graphene oxide grids with 2 nm gold fiducials for <3 Å resolution .
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Classification strategies: Focus 3D variability analysis on the F-box domain (residues 30-150). A recent study on SARS-CoV-2 antibodies achieved 2.8 Å resolution by masking non-RBD regions .
Epitope Mapping Workflow:
| Step | Key Parameter | Outcome |
|---|---|---|
| Antibody-antigen ratio | 3:1 (Antibody:At1g21990) | Saturates binding sites |
| Data Collection | 8,000 micrographs @ 300 kV | 60,000 particles |
| Post-processing | Focused refinement on residues 50-120 | Local resolution ≤3.5 Å |
What statistical approaches reconcile conflicting transcriptomic and proteomic data for At1g21990?
Leverage multi-omics integration:
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RNA-seq vs. TMT-MS: Apply linear mixed-effect models with batch effects as random factors. For Camelina sativa, a 1.5-fold mRNA-protein discordance is typical due to ubiquitination-mediated turnover .
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Dynamic simulations: Model protein half-life using ordinary differential equations. Example parameters:
Discordance Analysis Table:
| Condition | mRNA Fold Change | Protein Fold Change | p-value |
|---|---|---|---|
| Heat Stress | +3.2 | +1.1 | 0.003 |
| Drought | +2.1 | +0.9 | 0.021 |
How to design a phase II clinical trial for At1g21990-targeted therapeutics?
While no current trials target At1g21990, extrapolate from monoclonal antibody frameworks :
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Endpoint selection: Use progression-free survival (PFS) with a hazard ratio ≤0.6.
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Dose escalation: Apply a 3+3 design with 1 mg/kg, 3 mg/kg, and 10 mg/kg cohorts.
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PK/PD modeling: Estimate clearance rates using two-compartment models. For F-box inhibitors, target trough concentrations ≥5× in vitro IC90 .
Methodological Best Practices
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Antibody storage: Stabilize in PBS + 50% glycerol at -80°C; avoid freeze-thaw cycles >3× .
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In vivo dosing: For plant studies, infiltrate Agrobacterium strains (OD600 = 0.5) with 100 µM acetosyringone .
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Data transparency: Share raw SPR sensorgrams and cryo-EM density maps via public repositories (e.g., EMDB, PRIDE).
