rab-18 Antibody
Description
What is RAB18 Antibody?
RAB18 antibodies are polyclonal or monoclonal reagents that bind specifically to the RAB18 protein, a small GTPase involved in intracellular trafficking and organelle dynamics . These antibodies are widely used in techniques like Western blot (WB), immunohistochemistry (IHC), and immunofluorescence (IF) to study RAB18’s expression, localization, and function .
Applications in Research
RAB18 antibodies enable diverse experimental approaches:
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Western Blot: Detects RAB18 at ~23 kDa in human tissues (e.g., brain, liver) and cancer cell lines .
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Immunohistochemistry: Highlights cytoplasmic localization in prostate glandular cells and neuronal tissues .
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Functional Studies: Used to explore RAB18’s role in lipid droplet growth, ER-endosome contacts, and cancer metastasis .
Role in Cancer Progression
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Hepatocellular Carcinoma (HCC):
Neurological Disorders
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Warburg Micro Syndrome:
Lipid Metabolism
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Lipid Droplet (LD) Regulation:
Comparison of Select RAB18 Antibodies
Technical Considerations
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Validation: Reputable suppliers provide data showing clear bands at ~23 kDa in WB and distinct cytoplasmic staining in IHC .
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Storage: Lyophilized or liquid formulations stored at -20°C; avoid freeze-thaw cycles .
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Controls: Use tissues/cells with known RAB18 expression (e.g., HCC lines, brain samples) .
Product Specs
**Constituents:** 50% Glycerol, 0.01M PBS, pH 7.4
Target Background
Q&A
How to validate RAB18 antibody specificity in Western blot for diverse cellular models?
Methodological approach:
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Knockout controls: Use CRISPR/Cas9-generated RAB18-knockout cell lines (e.g., Hep3BCas9 cells) to confirm absence of bands at ~22 kDa ( ).
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Cross-species validation: Test antibody reactivity across human, mouse, and model organisms (e.g., Chlamydomonas) using lysates from cells expressing HA-tagged RAB18 variants ( ).
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Competition assays: Pre-incubate antibodies with recombinant RAB18 protein (1–10 µg/mL) to verify signal reduction.
Data interpretation:
| Validation Step | Expected Outcome | Common Pitfalls |
|---|---|---|
| Knockout lysate | No band at 22 kDa | Off-target binding to RAB1/3 isoforms |
| Tagged overexpression | Band shift matching tag size (e.g., +3 kDa for HA tag) | Endogenous vs. exogenous protein competition |
What experimental designs address conflicting reports on RAB18’s role in ER-LD tethering versus focal adhesion dynamics?
Advanced strategy:
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Conditional knockdown: Combine siRNA-mediated RAB18 silencing (e.g., siRAB18_3 at 20 nM ) with live-cell imaging of ER (CLIMP-63 markers) and focal adhesions (vinculin-GFP) in fibronectin-adherent cells ( ).
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Dose-response profiling: Treat cells with 6TG (100 µM ) to assess functional redundancy between RAB18 and ZW10/STX18 in survival assays.
Conflict resolution framework:
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Context dependency: Compare cancer vs. primary cells (e.g., Hep3B vs. fibroblasts ).
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Temporal analysis: Monitor RAB18 localization at 0, 15, 60 min post-stimulation with EGF (10 ng/mL).
How to optimize immunofluorescence protocols for RAB18 detection in polarized cells?
Technical guidelines:
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Fixation: Use ice-cold methanol (10 min) over paraformaldehyde to preserve ER-associated RAB18-GTP conformations ( ).
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Antibody dilution: Titrate anti-RAB18 (Abcam) between 1:150–1:300 in 5% BSA/TBST.
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Counterstains: Combine with KIF5B (1:100 ) for motor protein colocalization studies.
Quantitative thresholds:
| Parameter | Optimal Range |
|---|---|
| Exposure | 50–200 ms (488 nm channel) |
| Signal:Noise | >3:1 (vs. IgG control) |
What multi-omics approaches elucidate RAB18’s pleiotropic functions?
Integrated workflow:
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Proteomics: Immunoprecipitate RAB18 complexes using GFP-TRAP® beads, followed by LC-MS/MS (≥5 biological replicates ).
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Transcriptomics: Pair with single-cell RNA-seq in RAB18-KO models to identify HPRT1-like compensatory pathways (FDR <0.1 ).
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Metabolomics: Profile lipid droplets via MALDI-TOF in Rab18-depleted hepatocytes.
Key findings from literature:
| Interaction Partner | Functional Role | Technique | Source |
|---|---|---|---|
| Kinectin (KNT1) | ER-to-Golgi transport | Co-IP/WB | |
| ZW10/STX18 | LD biogenesis | CRISPR screen | |
| BBS8 | Ciliary signaling | YFP-tagging |
How to troubleshoot RAB18 antibody cross-reactivity in IHC for aging studies?
Solution framework:
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Pre-absorption: Incubate antibody with spleen lysates (1:10 w/v) to block non-specific IgGs common in aged tissue ( ).
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Age-stratified controls: Include samples from donors aged 18–96 with RIN >8.0 for RNA co-analysis ( ).
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Multiplex validation: Pair with γH2AX staining to distinguish true RAB18 signals from age-related autofluorescence.
Age-associated biomarkers:
| Biomarker | Correlation with RAB18 | Assay |
|---|---|---|
| CD8+ naïve T cells | r = -0.62 (p < 0.01) | Flow cytometry |
| Serum Eotaxin | r = +0.54 (p < 0.05) | ELISA |
What mechanistic studies resolve RAB18’s dual role in siRNA efficacy and vesicle trafficking?
Hypothesis-driven design:
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Trafficking blockade: Treat cells with Retro-2 (10 µM, 6 hr) to inhibit COPI-independent transport and measure GalNAc-HPRT1 siRNA IC50 shifts ( ).
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Structure-function analysis: Express GDP-locked (S22N) vs. GTP-bound (Q67L) RAB18 mutants in knockout lines ( ).
