At2g18190 Antibody
Description
Antibody Structure and Function
Antibodies are Y-shaped proteins composed of two heavy chains and two light chains connected by disulfide bonds. Each chain contains variable (antigen-binding) and constant regions, with the paratope region determining antigen specificity . In plant research, antibodies are used to detect proteins involved in defense responses, signaling pathways, or metabolic processes .
Antibody Applications in Arabidopsis Research
Antibodies targeting Arabidopsis proteins are critical for:
-
Immunoblotting: Detecting protein expression levels under stress conditions (e.g., RSV infection in Arabidopsis) .
-
Immunofluorescence: Localizing proteins within tissues or cells .
-
Functional Studies: Investigating roles of specific genes in defense mechanisms or development .
Example: Defense-Related Antibodies in Arabidopsis
While At2g18190 is not explicitly discussed, source highlights antibodies targeting Arabidopsis genes such as:
| Gene ID | Protein Function | Expression During RSV Infection |
|---|---|---|
| AT5g45000 | Disease resistance TIR-NBS-LRR | Upregulated at 14 dpi |
| AT4g03450 | Ankyrin repeat family protein | Upregulated at 14 dpi |
| AT5g10760 | EDS1-dependent 1 | Downregulated at 21 dpi |
These studies reveal dynamic antibody-detected changes in defense-related proteins during viral infection .
Challenges in Antibody Validation
Antibody reliability depends on rigorous characterization:
-
Specificity: Knockout cell lines are ideal controls to confirm target recognition .
-
Cross-Reactivity: Polyclonal antibodies may bind multiple epitopes, necessitating validation .
-
Reproducibility: Only ~50% of commercial antibodies perform as advertised in standard assays .
Future Directions
Emerging methods for antibody development include:
Product Specs
Composition: 50% Glycerol, 0.01M PBS, pH 7.4
Target Background
KEGG: ath:AT2G18190
STRING: 3702.AT2G18190.1
Q&A
Basic Research Questions
-
How do I validate the specificity of a custom antibody against a plant protein (e.g., At2g18190)?
-
Methodology:
-
Perform Western blotting using protein extracts from wild-type and knockout plant lines (e.g., Arabidopsis T-DNA mutants). Compare immunoreactive bands between samples to confirm target specificity .
-
Use peptide blocking assays: Pre-incubate the antibody with the antigenic peptide to verify signal loss .
-
Include orthogonal validation (e.g., immunoprecipitation followed by mass spectrometry) to confirm target identity .
-
-
-
What experimental controls are critical when using antibodies in plant tissue imaging?
Advanced Research Questions
-
How can computational tools improve antibody design for plant proteins with low solubility?
-
Approach:
-
Leverage automated pipelines like the CamSol-FoldX method to co-optimize conformational stability and solubility. Input the target protein’s structure to predict mutations that enhance developability without compromising binding .
-
Example workflow:
Step Tool/Resource Outcome 1. Structure modeling AlphaFold/Phyre2 Generate 3D model of At2g18190 2. Solubility prediction CamSol webserver Rank mutations improving solubility 3. Stability prediction Fold-X Assess ΔΔG of mutations
-
-
-
How do I resolve contradictory data between antibody-based assays and transcript/protein quantification methods?
-
Troubleshooting:
-
Scenario: Discrepancy between Western blot signal and RNA-seq/qRT-PCR data.
-
Solutions:
-
-
Key Considerations from Literature
-
Antibody Validation: Commercial antibodies often lack rigorous specificity testing. For example, AT2 receptor antibodies showed identical bands in wild-type and knockout mice, indicating nonspecific binding .
-
Automated Optimization: Computational methods can concurrently enhance solubility and stability, as demonstrated for therapeutic antibodies .
-
Mitochondrial Targets: Antibodies against subunits of complexes like ATP synthase require validation in functional assays (e.g., oligomycin sensitivity tests) .
