RAB18 Antibody

What Is RAB18 and Why Is It Important for Cell Biology Research?

RAB18 is a member of the RAS oncogene family, specifically belonging to the Rab protein subfamily of small GTPases. This 23 kDa protein (206 amino acids) plays crucial roles in multiple cellular functions:

• Regulation of endoplasmic reticulum (ER) transport toward the cell surface

• Focal adhesion dynamics during cell migration

• ER-lipid droplet (LD) contact formation

• Key involvement in eye and brain development

Research significance: RAB18 deficiency has been linked to Warburg micro syndrome type 3, a neurodevelopmental disorder. Additionally, recent studies have demonstrated RAB18's role in cancer progression, particularly in hepatocellular carcinoma where high expression correlates with poor prognosis .

In experimental models, RAB18 knockdown significantly impairs cell spreading and directional migration. For instance, studies have shown that cells silenced for RAB18 exhibit approximately 50% reduction in cell spreading area compared to control cells (control ~500 μm² vs. RAB18-silenced <300 μm²) .

What Are the Optimal Applications for RAB18 Antibodies in Research?

RAB18 antibodies have been validated for multiple applications, with varying success rates and recommended protocols:

For advanced applications: RAB18 antibodies have been successfully used in knockout/knockdown validation studies and co-immunoprecipitation experiments to identify binding partners like kinectin-1 (KNT1) and kinesin-1 (KIF5B) .

Methodological recommendation: Always titrate the antibody in your specific experimental system to determine optimal conditions, as antibody performance can vary significantly depending on tissue type and preparation methods .

How Should Western Blot Protocols Be Optimized for RAB18 Detection?

Successful Western blot detection of RAB18 requires specific optimization steps:

Sample preparation considerations:

• Include protease inhibitors to prevent degradation

• Use RIPA or NP-40 buffer for effective extraction from membrane compartments

• Load 10-30 μg of total protein for optimal detection

Recommended protocol modifications:

• Use 12-15% SDS-PAGE gels to effectively resolve the 20-23 kDa RAB18 protein

• Transfer at 100V for 60-90 minutes using PVDF membranes (preferred over nitrocellulose)

• Block with 5% non-fat milk in TBST for 1 hour at room temperature

• Incubate with primary antibody (1:1000-1:6000 dilution) overnight at 4°C

• Wash extensively (4 × 10 minutes) with TBST

• Use HRP-conjugated secondary antibodies at 1:5000 dilution

Troubleshooting guidance: If multiple bands appear, validate specificity using knockdown controls. The observed molecular weight range of 20-23 kDa reflects potential post-translational modifications of RAB18 .

What Sample Types Have Been Successfully Used with RAB18 Antibodies?

RAB18 antibodies have been validated across diverse tissue types and species:

Research findings demonstrate that RAB18 expression is ubiquitous but varies significantly between tissues. For instance, immunohistochemical assays have shown that RAB18 expression is significantly higher in hepatocellular carcinoma tissues (61.3%, 57/93 specimens) compared to adjacent non-tumor tissues (37.9%, 33/87) .

Methodological note: When working with new sample types, always include positive controls from validated tissues (e.g., brain tissue) to confirm antibody functionality .

How Do Different Fixation and Antigen Retrieval Methods Affect RAB18 Antibody Performance in Immunohistochemistry?

Fixation and antigen retrieval methods significantly impact RAB18 antibody performance in IHC applications:

Recommended fixation protocols:

• Formalin fixation (10% neutral buffered) for 24-48 hours is optimal for most tissues

• Freshly prepared paraformaldehyde (4%) for 15-20 minutes is preferred for cultured cells

Antigen retrieval comparison:

• TE buffer (pH 9.0): Primary recommendation for most tissue types; provides stronger signal with lower background

• Citrate buffer (pH 6.0): Alternative method that may work better for certain tissue types but often yields lower signal intensity

Research has shown that antigen retrieval in TE buffer pH 9.0 significantly improves detection in mouse brain and human cancer tissues compared to citrate buffer methods .

For optimal results in paraffin-embedded human prostate cancer tissue, studies have used 1:100 dilution after antigen retrieval, producing clear membrane and cytoplasmic staining patterns .

What Are the Critical Considerations for Co-localization Studies with RAB18 and Other Cellular Markers?

When designing co-localization experiments with RAB18 antibodies:

Recommended marker combinations:

• ER markers: Anti-CLIMP-63 (1:200), anti-RTN3 (1:1000) for ER subdomains

• Lipid droplet markers: BODIPY or anti-perilipin antibodies

• Focal adhesion markers: Anti-vinculin (1:150), anti-FAK and anti-pFAK (1:3000)

Technical considerations:

• Use sequential staining for rabbit polyclonal RAB18 antibodies to avoid cross-reactivity

• Select fluorophores with minimal spectral overlap (e.g., Alexa 488/Alexa 594/Alexa 647)

• Include no-primary-antibody controls for each secondary antibody

In advanced research settings, studies have successfully demonstrated RAB18 co-localization with kinectin-1 and kinesin-1 to reveal its role in ER transport mechanisms. For these studies, anti-KIF5B (1:100) and anti-KNT1 (1:150) antibodies were used in combination with anti-RAB18 antibodies .

How Can RAB18 Knockdown/Knockout Models Be Used to Validate Antibody Specificity?

Proper validation of RAB18 antibodies requires rigorous controls:

Recommended validation methods:

• siRNA-mediated knockdown: Use two different siRNA sequences targeting RAB18 (particularly 3'UTR-targeting siRNAs for rescue experiments)

• CRISPR/Cas9 knockout: Generate complete RAB18 knockout cell lines for absolute negative controls

• Rescue experiments: Re-express siRNA-resistant RAB18 to confirm specificity of observed phenotypes

Published validation approach:
Research has demonstrated that cells treated with RAB18-specific siRNAs show significant reduction in Western blot signal at 20-23 kDa. In these studies, transfection with GFP-tagged RAB18 resistant to siRNA restored the signal, confirming antibody specificity .

Quantitative assessment showed that depletion of RAB18 using specific siRNAs resulted in:

• Altered distribution of ER markers (e.g., CLIMP-63)

• Reduced cell spreading by approximately 50%

• Disrupted directional migration in chemotaxis assays

What Methodological Approaches Are Effective for Studying RAB18's Role in Focal Adhesion Dynamics?

To investigate RAB18's involvement in focal adhesion (FA) dynamics:

Recommended experimental design:

• Focal adhesion visualization: Use anti-vinculin antibodies (1:150 dilution) to identify FAs

• Dynamic studies: Employ GFP-paxillin or GFP-vinculin for live-cell imaging

• Micropattern assays: Utilize L-shaped fibronectin-coated patterns to normalize cell shape and quantify cytoskeletal organization

Quantification methods:

• Measure FA size, number, and distribution using ImageJ/FIJI software

• Calculate FA assembly/disassembly rates through time-lapse imaging

Research findings have demonstrated that RAB18 knockdown decreases the total number of FAs without affecting the number of FAs per cell area, suggesting a defect in cell spreading. Additionally, cells on L-shaped micropatterns showed approximately 50-70% reduction in full spreading capability when RAB18 was silenced .

For advanced studies, researchers have successfully used RAB18 antibodies in combination with phospho-FAK (Tyr397) antibodies to assess FA maturation and turnover .

How Do Different Rab18 Antibody Clones Compare in Terms of Specificity and Applications?

Selection of the appropriate RAB18 antibody clone is critical for experimental success:

Performance comparison:
Research has shown variable performance between clones. For instance, polyclonal antibody 11304-1-AP has been successfully used in detecting RAB18 in multiple tissues including brain, heart, and testis tissues from human, mouse and rat samples .

For validation studies involving protein-protein interactions, antibodies against specific binding partners have been used at defined dilutions: anti-KIF5B (1:1000 for WB, 1:100 for coIP) and anti-KNT1 (1:2000 for WB, 1:50 for coIP) .

What Are the Key Technical Considerations When Using RAB18 Antibodies in Co-immunoprecipitation Studies?

For successful co-immunoprecipitation (coIP) studies with RAB18 antibodies:

Optimized protocol:

• Lyse cells in mild detergent buffer (e.g., 1% NP-40, 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 1 mM EDTA with protease inhibitors)

• Pre-clear lysates with protein A/G beads for 1 hour at 4°C

• Incubate cleared lysates with RAB18 antibody (0.5-4.0 μg for 1.0-3.0 mg total protein) overnight at 4°C

• Add fresh protein A/G beads for 1-2 hours at 4°C

• Wash extensively (4-5 times) with lysis buffer

• Elute bound proteins by boiling in SDS sample buffer

Research applications:
CoIP studies have successfully demonstrated that RAB18 interacts with kinectin-1 (KNT1), specifically binding to the cytoplasmic region between residues 200 and 445 of KNT1. These interactions have been validated through both overexpression studies and endogenous protein IP experiments .

Direct protein-protein interactions have been confirmed using purified recombinant proteins. For instance, GST-tagged KNT1 (residues 201-445) was shown to directly bind His-tagged RAB18 Q67L in pull-down assays, providing a powerful approach for validating antibody-based findings .

How Can Rab18 Antibodies Be Used to Investigate Its Role in Lipid Droplet Dynamics?

For studying RAB18's functions in lipid droplet (LD) biology:

Experimental approach:

• Induce LD formation with oleic acid treatment (typically 0.25-0.5 mM for 1-24 hours)

• Co-stain LDs with BODIPY 493/503 (1 μg/ml) or LipidTOX

• Use RAB18 antibodies at 1:200 dilution for immunofluorescence

• Analyze LD size, number, and distribution

Key findings from published research:
EM analysis has revealed that RAB18-deficient cells show dramatic changes in LD morphology. After 8 hours of oleic acid treatment, control cells develop LDs larger than 150 nm in diameter, while RAB18-deficient cells predominantly maintain LDs smaller than 150 nm (except for a few LDs larger than 2 μm) .

Quantitatively, RAB18-deficient cells show:

• 88% reduction in mature LD numbers (117 ± 13 per cell vs. 938 ± 105 in control cells)

• Significant increase in supersized LDs

• Nearly two-fold larger diameter of the largest LD (representing an eight-fold volume increase)

What Controls Should Be Included When Using RAB18 Antibodies in Different Experimental Systems?

To ensure scientific rigor when working with RAB18 antibodies:

Essential control panel:

Implementation in published research:
Studies have demonstrated that reintroducing GFP-RAB18 into RAB18-silenced cells rescues observed phenotypes, providing strong validation of antibody specificity. For example, expression of GFP-RAB18 in cells silenced for RAB18 rescued spreading defects and restored normal chemotaxis orientation .