At3g25550 Antibody
Description
Introduction to At3g25550 Antibody
The At3g25550 antibody is a polyclonal antibody raised against the At3g25550 protein in Arabidopsis thaliana (mouse-ear cress) . Antibodies are Y-shaped proteins produced by the immune system to identify and neutralize foreign invaders such as bacteria and viruses . At3g25550 is a gene name in Arabidopsis thaliana that codes for a probable F-box protein .
1.1. Basic Antibody Structure
Antibodies, also known as immunoglobulins (Igs), are glycoproteins produced by B lymphocytes . A typical antibody molecule has a Y-shape and a molecular weight of approximately 150 kDa . The basic structure includes two identical halves, each consisting of a heavy chain (~50 kDa) and a light chain (~25 kDa) linked by disulfide bonds . The heavy chain comprises an Fc (fragment crystallizable) region at the carboxyl terminal and a Fab (fragment antigen-binding) region at the amino terminal . The Fab region binds to antigens, while the Fc region interacts with immune cells to trigger immune responses .
Structure and Isotypes
Antibodies have two light chain types: lambda (λ) and kappa (κ), and five heavy chain isotypes: μ, δ, γ, α, and ε . The structural differences in the constant region of antibodies determine their class and divide them into five categories .
2.1. IgG Antibody
IgG is a tetrameric molecule with two light chains (25 kDa) and two gamma (γ) heavy chains (50 kDa) . It participates in various immune responses, including activating the complement system and neutralizing bacterial toxins, and can cross the placenta . IgG is the most abundant antibody in the body, with a molecular weight of 150,000 Da (150 kDa), making up about 80% of serum immunoglobulin and has a half-life of 23 days . IgG has two gamma (γ) heavy chains and two lambda (λ) or kappa (k) light chains, providing two identical antigen-binding sites and is further divided into four subclasses: IgG1, IgG2, IgG3, and IgG4, based on minor differences in their heavy chains .
Research Applications of Antibodies
Antibodies are essential tools in biological research and diagnostics, with various applications, including:
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Western blotting: Antibodies are used to detect specific proteins in a sample .
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ELISA (enzyme-linked immunosorbent assay): They quantify the amount of a protein in a sample .
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Immunofluorescence microscopy: Used to visualize the location of a protein within cells or tissues .
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Therapeutics: Antibodies can be engineered to target specific cells or proteins for therapeutic purposes, such as in cancer therapy or to reduce tau proteins found in the brains and retinas of mice with Alzheimer's disease .
At3g25550 Protein Function
The At3g25550 gene in Arabidopsis thaliana encodes a probable F-box protein . F-box proteins are components of the SCF (Skp1-Cullin-F-box) ubiquitin ligase complexes, which are involved in regulating various cellular processes, including signal transduction, cell cycle progression, and transcriptional regulation .
Table: Antibody Isotypes and Characteristics
| Isotype | Heavy Chain | Molecular Weight (kDa) | Percentage of Serum Ig | Half-Life (days) | Function |
|---|---|---|---|---|---|
| IgG | Gamma (γ) | 150 | 80% | 23 | Neutralizes toxins, opsonization, complement activation, crosses placenta |
| IgM | Mu (μ) | 970 | 5-10% | 5 | Activates complement, first antibody produced during an immune response |
| IgA | Alpha (α) | 160 (monomer), 400 (dimer) | 10-15% | 6 | Mucosal immunity, found in secretions (saliva, milk) |
| IgE | Epsilon (ε) | 190 | <0.002% | 2 | Involved in allergic reactions, binds to mast cells and basophils |
| IgD | Delta (δ) | 175 | <1% | 1-3 | Expressed on mature B cells, function not fully understood, may play a role in B cell activation and differentiation |
Product Specs
Q&A
How can researchers validate the specificity of the At3g25550 antibody in Arabidopsis thaliana experiments?
To confirm antibody specificity, a multi-modal approach is essential. First, perform Western blotting using protein extracts from wild-type Arabidopsis and at3g25550 knockout mutants. A specific antibody will produce a band at the expected molecular weight (~X kDa) in wild-type samples and show no signal in knockout lines . Second, use immunocytochemistry in mutant vs. wild-type root/shoot tissues to verify spatial localization patterns. Third, pre-absorb the antibody with recombinant At3g25550 protein; binding signals should diminish proportionally to antigen concentration. For quantitative validation, combine these results with mass spectrometry-based proteomic profiling of immunoprecipitated samples to confirm target enrichment.
What are the optimal tissue preparation protocols for At3g25550 detection in plant systems?
Preserving epitope integrity requires tissue-specific protocols:
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Root tissues: Fix in 4% paraformaldehyde for 20 min, then permeabilize with 0.1% Triton X-100.
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Leaf tissues: Vacuum-infiltrate with PBS containing 1 mM EDTA to maintain membrane stability.
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Seeds: Use cryosectioning after embedding in OCT compound to overcome lipid-rich barriers.
Include controls with competing peptides to distinguish non-specific background . For phosphorylation-state studies, add phosphatase inhibitors (e.g., 1 mM Na3VO4) during extraction.
How should researchers design experiments to investigate At3g25550’s role in protein-protein interaction networks?
Adopt a triangulation strategy:
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Co-immunoprecipitation (Co-IP): Crosslink interacting partners with 1% formaldehyde before extraction, using IgG isotype controls to exclude non-specific binding .
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Bimolecular fluorescence complementation (BiFC): Fuse At3g25550 to the N-terminal fragment of YFP and candidate interactors to the C-terminal fragment. Quantify reconstituted fluorescence via confocal microscopy.
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Structural modeling: Generate homology models using AlphaFold2 to predict interaction interfaces, then validate through site-directed mutagenesis of residues (e.g., D127A, K189E) that disrupt binding.
Table 1: Comparison of Protein Interaction Validation Methods
| Method | Resolution | Throughput | False Positive Rate | Key Controls Required |
|---|---|---|---|---|
| Co-IP | 5–10 nm | Low | 15–20% | Isotype control, competition assay |
| BiFC | 10–50 nm | Medium | 10–15% | Non-interacting partner pair |
| FRET-FLIM | 1–5 nm | High | 5–8% | Donor-only acceptor control |
What computational approaches resolve contradictions in At3g25550 subcellular localization data?
Discrepancies between studies often arise from dynamic relocalization or fixation artifacts. Implement:
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Live-cell imaging: Tag At3g25550 with pH-sensitive GFP variants (e.g., pHluorin) to monitor real-time trafficking between compartments like the plasma membrane and vacuole.
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Machine learning-based image analysis: Train U-Net models on ground-truth datasets to distinguish true signal from autofluorescence in chloroplast-containing tissues .
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Orthogonal validation: Compare results across ≥3 methods (e.g., immunogold EM, fractionation proteomics, and fluorescent reporter lines).
How can researchers optimize the At3g25550 antibody for single-cell RNA sequencing (scRNA-seq) compatibility?
Traditional antibodies may interfere with transcriptome amplification. Use:
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Barcode-conjugated antibodies: Link At3g25550-specific Fab fragments to oligonucleotide barcodes detectable in 10x Genomics workflows.
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Cross-validation pipeline:
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Perform scRNA-seq on protoplasts from antibody-treated vs. untreated plants.
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Cluster cells using Seurat and confirm protein expression correlates with At3g25550 mRNA levels (expected Pearson’s r > 0.7).
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Apply CITE-seq controls with hashed antibody tags to quantify non-specific binding.
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What statistical frameworks are robust for analyzing At3g25550 knockout phenotype variability?
Address heterogeneity through:
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Mixed-effects modeling: Account for random effects like batch variation and plant growth chamber microenvironments.
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Bayesian hierarchical models: Estimate posterior probabilities of phenotype severity across tissue types.
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Power analysis: For RNA-seq experiments, ensure n ≥ 6 biological replicates to detect 1.5-fold expression changes (α = 0.05, power = 0.8).
