FIM5 Antibody
Description
Definition and Biological Context
FIM5 (FIMBRIN5) is an actin-binding protein critical for organizing apical actin filaments in Arabidopsis thaliana pollen tubes. The FIM5 Antibody is a research tool used to study its localization, function, and role in polarized cell growth. It is primarily applied in immunolocalization experiments to visualize FIM5’s interaction with actin structures during pollen tube development .
Role in Pollen Tube Growth
FIM5 stabilizes apical actin filaments, ensuring proper growth and morphology. Key findings include:
Actin Organization
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Wild-Type Pollen Tubes: FIM5 decorates actin filaments throughout the tube, with apical concentration. Actin fringes and longitudinal bundles are well-organized .
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fim5 Mutants:
Quantitative Actin Parameters
| Parameter | Wild-Type (Early Stage) | fim5 Mutant (Early Stage) |
|---|---|---|
| Actin Convolutedness | Baseline | ↑ 2.3× (p < 0.01) |
| Actin Bundle Density | High | ↓ 40% |
| Growth Rate | 0.8–1.2 μm/min | ↓ 30–50% |
Antibody Development and Applications
The FIM5 Antibody is typically generated via recombinant protein expression and purification:
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Antigen Production:
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Antibody Purification:
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Validation:
Functional Disruption via Antibody Microinjection
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Lily Pollen Tubes: Microinjection of anti-FIM1 (homologous to FIM5) antibodies inhibits tip growth and disrupts actin fringes, mirroring fim5 mutant phenotypes .
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Mechanistic Insights:
Implications for Plant Biology
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Actin Dynamics: FIM5’s role in bundling and rigidity regulation highlights conserved mechanisms in tip-growing cells .
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Agricultural Applications: Insights into FIM5 function could inform strategies to enhance pollen fertility or resilience .
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Cross-Species Relevance: Homologs like Ll-FIM1 in lily demonstrate evolutionary conservation of FIMBRIN function .
Limitations and Future Directions
Product Specs
Constituents: 50% Glycerol, 0.01M PBS, pH 7.4
Target Background
FIM5 Function: Research indicates FIM5 plays a crucial role in actin organization and dynamics, particularly within plant pollen grains and pollen tubes. Specific functions include:
- Organization and maintenance of normal actin architecture in pollen grains and pollen tubes, essential for double fertilization. (PMID: 29036437)
- Decoration of apical actin filaments and regulation of their organization to drive polarized pollen tube growth. (PMID: 27117336)
- In contrast to FIM4, which generates both actin bundles and cross-linked actin filaments, FIM5 specifically generates actin bundles. This functional distinction is evident at the single-filament level. (PMID: 27261463)
- Regulation of actin dynamics and organization during pollen germination and tube growth by stabilizing actin filaments and organizing them into higher-order structures. (PMID: 21098731)
Q&A
Given the lack of specific information on "FIM5 Antibody" in the search results, I will focus on creating a collection of FAQs that are relevant to antibody research in general, while ensuring they meet the requirements for depth, distinction between basic and advanced questions, and methodological answers. Since "FIM5" is not directly referenced in the provided search results, I will use the term as a placeholder for a hypothetical antibody and draw from broader antibody research principles.
Answer:
To validate the specificity of an antibody in IF assays, ensure that the antibody detects the correct subcellular localization of the target protein. Use a combination of positive and negative controls, such as cells expressing the target protein and those lacking it. Optimize fixation and permeabilization conditions to enhance specificity and consistency .
Answer:
When discrepancies arise between predicted and measured antibody activity, re-evaluate the assumptions of your model, such as independent or exclusive binding. Consider analyzing fewer antibodies to simplify the model or incorporating additional data on pairwise interactions and epitope mapping . Validate the model with experimental data to ensure accuracy.
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To determine the epitope specificity of an antibody, use techniques such as peptide array analysis or competition assays with known epitope-specific antibodies. These methods help identify the specific region on the antigen that the antibody binds to, which is crucial for understanding its behavior in complex mixtures .
Answer:
When characterizing an antibody for multiple applications, consider its specificity, sensitivity, and consistency across different assays. Ensure that the antibody performs well in each application by validating it with appropriate controls and optimizing assay conditions. This includes verifying the correct subcellular localization in IF and detecting the expected bands in Western Blot .
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Common issues with antibodies include non-specific binding, inconsistent performance, and lack of specificity. These can be addressed by optimizing assay conditions, using appropriate controls, and ensuring lot-to-lot consistency. Additionally, rigorous validation processes, such as those used by Cell Signaling Technology, can help mitigate these issues .
Answer:
Data mining can accelerate antibody discovery by analyzing large datasets of antibody sequences to identify modifications that reduce immunogenicity or enhance therapeutic properties. Combining public and proprietary data can help engineer antibodies with improved specificity and efficacy .
Answer:
Full sequence validation of therapeutic antibodies is crucial for ensuring their integrity and efficacy. Techniques such as middle-up and middle-down mass spectrometry are used to confirm the reference sequences of antibodies. This validation is essential for regulatory compliance and to ensure the consistency of biotherapeutic products .
Answer:
To address the antibody characterization crisis, researchers should prioritize rigorous validation of antibodies using multiple assays and controls. Initiatives like NeuroMab provide strategies for generating and validating high-quality antibodies. Additionally, journals and funding agencies should enforce strict standards for antibody characterization in published research .
Example Data Table: Antibody Validation Methods
| Assay | Purpose | Controls Needed |
|---|---|---|
| Western Blot | Specificity and Sensitivity | Positive and Negative Controls |
| Immunofluorescence | Subcellular Localization | Cells with and without target protein |
| ELISA | Quantification and Specificity | Known positive and negative samples |
This table illustrates the importance of using multiple assays to comprehensively validate an antibody's performance.
Detailed Research Findings:
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Antibody Specificity: Ensuring an antibody binds specifically to its target is crucial. This can be verified through Western Blot by detecting a single band corresponding to the target protein .
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Data Mining in Antibody Engineering: Analyzing large datasets of antibody sequences can help identify modifications that improve therapeutic properties, such as reduced immunogenicity .
