KIN14E Antibody
Product Specs
Constituents: 50% Glycerol, 0.01M PBS, pH 7.4
Target Background
- Recent research has revealed a novel genetic and molecular mechanism where TCS1 interacts with KCBP to regulate trichome cell shape by influencing the stability of microtubules. PMID: 27768706
- The MyTH4 domain and the FERM domain located in the N-terminal tail of KCBP physically bind to microtubules and F-actin, respectively. PMID: 26287478
Q&A
Here’s a structured collection of FAQs for researchers working with KIN14E Antibody, designed to address both foundational and advanced scientific inquiries:
How to validate antibody specificity for KIN14E in plant systems?
Methodological Approach:
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Genetic controls: Use Arabidopsis thaliana knockout lines (e.g., zwichel mutants) to confirm absence of signal in negative controls .
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Orthogonal validation: Pair antibody staining with fluorescently tagged KIN14E constructs in transgenic lines .
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Cross-reactivity checks: Test specificity across related species (e.g., Brassica napus, Populus trichocarpa) using Western blotting .
Key Data:
| Validation Strategy | Example Application | Source |
|---|---|---|
| Knockout validation | Trichome morphogenesis assays | |
| Immunoprecipitation-MS | Protein interaction studies |
What experimental conditions optimize KIN14E immunolocalization in trichomes?
Protocol Recommendations:
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Fixation: Use 4% paraformaldehyde + 0.1% glutaraldehyde to preserve microtubule-cytoskeleton interactions .
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Permeabilization: 0.5% Triton X-100 for 15 min to balance membrane integrity and antibody penetration .
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Antibody dilution: Start at 1:500 in blocking buffer (5% BSA + 0.1% Tween-20).
Troubleshooting:
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Non-specific signals in root tissues? Pre-adsorb antibody with recombinant KIN14E (amino acids 861–1321) .
How does KIN14E’s motor activity regulate microtubule stability in vivo?
Mechanistic Insights:
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ATPase assays: Measure basal vs. microtubule-stimulated ATP hydrolysis rates (kin14-VIb: ~0.6 µm/s motility) .
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Liposome motility assays: Reconstitute KIN14E clusters on synthetic membranes to quantify retrograde transport processivity .
Critical Data:
Resolving contradictions in KIN14E’s role in root growth regulation
Analysis Framework:
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Genetic epistasis: Combine KIN14E RNAi with microtubule-disrupting drugs (e.g., oryzalin) to test additive effects.
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Live-cell imaging: Track EB1-GFP comets in root epidermal cells to quantify microtubule dynamics .
Case Study:
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Observation: KIN14E overexpression inhibits root growth but enhances trichome branching .
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Resolution: Tissue-specific promoters (e.g., GL2 for trichomes) clarify context-dependent roles .
Designing studies to explore KIN14E-TCS1 interactions
Experimental Pipeline:
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Co-IP/MS: Immunoprecipitate TCS1-GFP and probe for endogenous KIN14E .
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FRET-FLIM: Quantify interaction efficiency in trichome tip cells.
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Mutant analysis: Compare microtubule bundling in tcs1 vs. kin14e double mutants .
Key Finding:
TCS1 stabilizes KIN14E’s binding to microtubule minus ends, reducing catastrophe frequency by ~40% .
