rab18b Antibody

What is RAB18 and why is it significant in cellular research?

RAB18 is a highly conserved member of the Ras-related small GTPase family that regulates intracellular membrane trafficking. Its significance stems from multiple critical cellular functions:

• Regulates vesicle transport between the endoplasmic reticulum (ER) and Golgi apparatus

• Controls endocytosis/recycling pathways at the apical membrane

• Maintains ER structure and forms ER-lipid droplet contacts

• Plays essential roles in eye and brain development

• Implicated in various pathological conditions including neurodegeneration and cancer

Mutations in RAB18 are associated with Warburg Micro syndrome type 3, a rare autosomal recessive multisystem disorder characterized by ocular and neurological abnormalities .

Are there specific RAB18B antibodies available, and how do they differ from general RAB18 antibodies?

Based on current research literature, there are no commercially available antibodies specifically targeting a RAB18B isoform. The existing antibodies recognize the canonical RAB18 protein. While some Rab proteins have multiple isoforms (like RAB1A and RAB1B mentioned in search result ), current literature does not clearly define a distinct RAB18B isoform. Researchers should be aware that commercial antibodies labeled as "RAB18" target the protein encoded by the RAB18 gene (UNIPROT ID: Q9NP72) .

What types of RAB18 antibodies are available and what applications are they validated for?

Multiple types of RAB18 antibodies are available with different validation profiles:

Most antibodies have been validated for Western blot at dilutions ranging from 1:200-1:10000, IHC at 1:20-1:2000, and IP at 1:200-1:1000 . The observed molecular weight of RAB18 in Western blot applications is typically 20-23 kDa .

What are the optimal conditions for using RAB18 antibodies in immunofluorescence experiments?

For optimal RAB18 immunofluorescence staining:

• Fixation: 4% paraformaldehyde (PFA) in PBS for 15 minutes at room temperature

• Permeabilization: 0.1% Triton X-100 in PBS for 10 minutes at room temperature

• Blocking: PBS containing 5% BSA, 5% goat serum, and 0.01% Triton X-100 for 30 minutes at room temperature

• Primary antibody incubation: Dilute RAB18 antibody in blocking buffer at 1:50-1:800 (depending on specific antibody) and incubate overnight at 4°C

• Secondary antibody: Use appropriate species-specific Alexa Fluor-conjugated secondary antibodies (typically at 1:200-1:500 dilution)

• Co-localization markers: For accurate subcellular localization, co-stain with markers for ER (CLIMP-63), Golgi (GM130), or lipid droplets depending on your research focus

Research shows RAB18 typically localizes to the perinuclear region (Golgi) and ER membranes, with localization patterns changing based on its activation state .

How should I design knockout validation experiments for RAB18 antibodies?

Knockout validation is essential for confirming antibody specificity:

• siRNA knockdown approach:

  • Use multiple siRNA sequences targeting different regions of RAB18 mRNA

  • Include a non-targeting siRNA control

  • Validate knockdown efficiency by qPCR and Western blot

  • Confirm phenotypic effects match published observations (altered ER structure, reduced cell spreading)

• CRISPR-Cas9 knockout approach:

  • Generate stable RAB18 knockout cell lines

  • Validate gene disruption by sequencing

  • Confirm protein loss by Western blot with multiple antibodies

  • Create mosaic cultures of wildtype and knockout cells for direct comparison in the same field of view

• Rescue experiments:

  • Re-express siRNA-resistant RAB18 constructs in knockdown cells

  • Confirm restoration of normal RAB18 localization and function

  • Example: "Depletion of Rab18 using the Rab18.8 3′UTR duplex resulted in the spread of CLIMP-63 into the peripheral region, and this was reversed by expression of GFP-Rab18 resistant to the siRNA"

What is the recommended protocol for RAB18 immunoprecipitation experiments?

For successful RAB18 immunoprecipitation:

• Lysate preparation:

  • Use RIPA buffer supplemented with protease inhibitors

  • For mouse testis tissue, use approximately 4000μg total protein lysate

  • For cell lines, use 1-3mg total protein lysate per reaction

• Antibody amount:

  • Use 0.5-4.0μg antibody per IP reaction, with 3μg being optimal for tissue samples

  • Pre-clear lysate with protein A/G beads before adding antibody

• Detection:

  • For detection in Western blot after IP, use the same antibody at 1:300 dilution

  • Include appropriate controls (IgG control, input sample)

• Verification:

  • Validate by MS analysis or reverse IP with interacting partners

  • Confirm interaction with known partners (e.g., RAB3GAP complex, kinectin-1)

How do I distinguish between active (GTP-bound) and inactive (GDP-bound) RAB18 in my experiments?

Standard RAB18 antibodies detect total RAB18 regardless of nucleotide-binding state. To specifically study active RAB18:

• GTP-locked mutants: Generate RAB18 Q67L (constitutively active) and S22N (dominant negative) mutants for overexpression studies

• GST-pulldown assays: Use GST-tagged effector proteins that specifically bind GTP-bound RAB18 to isolate the active form from cell lysates

• Subcellular localization: Active RAB18 is predominantly membrane-associated, while inactive RAB18 is more cytosolic

  • "In cell lines where TBC1D20 is absent from the endoplasmic reticulum (ER), RAB18 becomes more stably ER-associated and less cytosolic than in control cells"

• Fluorescence recovery:

  • The fluorescence recovery profile differs between active and inactive forms

  • "The fluorescence recovery profile of RAB18 was significantly different in control and TBC1D20(p.Gln98*) cells"

What could cause unexpected bands or non-specific staining when using RAB18 antibodies?

Several factors may contribute to non-specific signals:

• Cross-reactivity with related Rab proteins:

  • RAB18 shares sequence homology with other Rab family members

  • Validate specificity with RAB18 knockout/knockdown controls

• Post-translational modifications:

  • RAB18 undergoes prenylation and GTP/GDP cycling

  • Different forms may show slight shifts in molecular weight or multiple bands

• Degradation products:

  • Include protease inhibitors in sample preparation

  • Avoid freeze-thaw cycles of antibodies

• Technical considerations:

  • Optimize antibody dilution (start with manufacturer recommendations)

  • Increase blocking time/concentration to reduce background

  • For tissues with high endogenous biotin, use biotin-blocking steps prior to primary antibody incubation

• Solution: Compare multiple anti-RAB18 antibodies raised against different epitopes to confirm specificity .

When investigating RAB18's role in disease models, what control experiments are essential?

When studying RAB18 in disease contexts:

• Expression levels:

  • Quantify RAB18 at both mRNA and protein levels

  • "Notable increased RAB18 expression" has been observed in glioma tissues

  • Compare with appropriate non-diseased controls from the same tissue type

• Functional assays:

  • For cancer studies, assess proliferation, apoptosis, and migration

  • "Rab18 silencing markedly suppressed cell proliferation and the expression of Ki67 and PCNA"

  • For neurological conditions, examine ER structure and neuronal morphology

• Rescue experiments:

  • Re-express wild-type RAB18 in knockdown/knockout cells to confirm specificity

  • Test disease-associated mutations (e.g., those found in Warburg Micro syndrome)

  • "Expression of GFP-Rab18 in cells silenced for Rab18 rescued the spreading defect"

• Pharmacological interventions:

  • Use drugs that affect RAB18 regulators (GEFs/GAPs)

  • For cancer studies, combine with standard therapies

  • "RAB18 silencing induced low cell viability as compared to siRNA-NC group" when treated with TMZ in glioma cells

How can I study RAB18's interaction with its regulatory proteins (GEFs and GAPs)?

To investigate RAB18's regulatory network:

• GEF interaction studies:

  • The RAB3GAP complex (RAB3GAP1/RAB3GAP2) acts as a GEF for RAB18

  • "When Rab3GAP2 was ectopically targeted to mitochondria, its expression triggered recruitment of both coexpressed Rab3GAP1 and Rab18 to this compartment"

  • Disease-causing mutations in RAB3GAP1 (T18P, E24V) and RAB3GAP2 (R426C) abolish GEF activity toward RAB18

• GAP interaction studies:

  • TBC1D20 functions as a GAP for RAB18

  • "In cell lines where TBC1D20 is absent from the endoplasmic reticulum (ER), RAB18 becomes more stably ER-associated"

  • Loss of TBC1D20 alters RAB18 localization and dynamics

• Nucleotide binding assays:

  • Measure GTP binding/hydrolysis rates using purified components

  • Compare wild-type RAB18 with disease-associated mutants

• Structural biology approaches:

  • Use X-ray crystallography or cryo-EM to analyze complexes

  • Focus on interaction interfaces for drug development

What methods can be used to investigate RAB18's role in cancer progression?

For cancer research applications:

• Expression analysis in patient samples:

  • Perform IHC on tissue microarrays

  • Compare RAB18 levels between tumor and adjacent normal tissues

  • "Glioma cells expressed high RAB18" compared to normal astrocytes

• Functional studies in cancer cells:

  • siRNA knockdown affects proliferation and apoptosis

  • "Silencing of RAB18 significantly induced a decrease in cell proliferation"

  • "A consequent increase in the cell apoptosis was observed upon Rab18 silencing"

• Drug sensitivity modulation:

  • RAB18 silencing enhances chemosensitivity

  • "Rab18 silencing could enhance TMZ sensitivity of glioma cells in time-dependent manner"

• Protein interaction networks:

  • RAB18 interacts with cancer-relevant proteins

  • "Rab18 interacted with V-set and immunoglobulin domain-containing 4 (VSIG4)"

  • This interaction may be critical for cancer progression

• In vivo models:

  • Generate xenograft models with RAB18 knockdown/overexpression

  • Assess tumor growth, metastasis, and response to therapy

How does RAB18 contribute to ER structure and focal adhesion dynamics?

RAB18's role in cellular architecture involves:

• ER structure maintenance:

  • RAB18 depletion alters ER distribution

  • "Depletion of Rab18 using the Rab18.8 3′UTR duplex resulted in the spread of CLIMP-63 into the peripheral region"

  • This effect is reversed by expressing siRNA-resistant RAB18

• Focal adhesion regulation:

  • "Knockdown of Rab18 reduces the size of focal adhesions (FAs) and influences their dynamics"

  • "Rab18 regulates kinectin-1 transport toward the cell surface to form ER–FA contacts, thus promoting FA growth and cell migration during chemotaxis"

• Cell spreading and migration:

  • RAB18 is essential for normal cell spreading

  • "The average area of control cells seeded and allowed to adhere on fibronectin-coated coverslips for 1 h was ∼500 µm², whereas the area of cells silenced for Rab18 was <300 µm², showing a significant reduction in cell spreading by ∼50%"

  • Micropattern experiments show RAB18's role in cytoskeletal organization

• Molecular mechanism:

  • RAB18 directly interacts with kinectin-1, an ER-resident protein

  • This interaction controls "the anterograde kinesin-1–dependent transport of the ER required for the maturation of nascent FAs and protrusion orientation toward a chemoattractant"

What are the best practices for validating a new RAB18 antibody in my experimental system?

A comprehensive validation approach should include:

• Expression systems:

  • Test in RAB18-overexpressing and knockdown/knockout systems

  • Compare multiple antibodies targeting different epitopes

• Multiple techniques:

  • Validate across Western blot, IHC, IF, and IP applications

  • Use standardized protocols for each application

• Mosaic approach for imaging:

  • "HAP1 WT and the HAP1 KO cell lines were labelled with different fluorescent dyes"

  • "WT and KO cells were plated in a 96-well plate as a mosaic"

  • Image both cell types in the same field of view

• Quantification:

  • "Quantification of immunofluorescence intensity hundreds of WT and KO cells was performed for each antibody tested"

  • Compare signal-to-noise ratios between positive and negative samples

• Control tissues/cells:

  • Test tissues known to express RAB18 (brain, testis, heart)

  • Include tissues from knockout mouse models if available

How does the sequence homology between RAB18 and other Rab proteins affect antibody specificity?

Addressing homology concerns:

• Sequence similarity issues:

  • RAB18 belongs to the large Rab GTPase family (>60 members in humans)

  • Shares structural features with other Rabs, particularly in the GTP-binding domains

  • The C-terminal hypervariable region offers greater uniqueness for antibody targeting

• Epitope selection:

  • Antibodies raised against full-length RAB18 may have higher cross-reactivity risk

  • Peptide antibodies targeting unique regions improve specificity

  • Compare antibodies generated against different epitopes (e.g., N-terminal vs. C-terminal)

• Validation requirements:

  • Knockout/knockdown controls are essential to confirm specificity

  • Test in tissues with varying expression patterns of different Rab proteins

  • Compare immunoblotting against purified recombinant Rab proteins

• Common cross-reactivity issues:

  • Some RAB18 antibodies may cross-react with related small GTPases

  • Always validate with appropriate controls

  • Consult literature for known cross-reactivity issues with specific antibodies

What information should be included when reporting RAB18 antibody usage in publications?

To ensure reproducibility, include:

• Complete antibody information:

  • Manufacturer, catalog number, lot number, RRID

  • Host species, clonality (monoclonal/polyclonal)

  • Target epitope information if available

• Validation data:

  • How specificity was confirmed (knockout, knockdown, etc.)

  • Known limitations or cross-reactivity

  • Representative images of controls

• Detailed protocols:

  • Sample preparation methods

  • Exact dilutions used for each application

  • Incubation times and temperatures

  • Antigen retrieval methods for IHC

  • Blocking reagents and conditions

  • Secondary antibody details

• Examples from literature:

  • "Primary antibodies used were anti-Rab18 (Abcam, 1:200), anti-Rab18 for GFP-TRAP experiments (Sigma-Aldrich 1:200)"

  • "Immunohistochemical of paraffin-embedded human prostate cancer using Catalog No:114415(RAB18 antibody) at dilution of 1:100 (under 40x lens)"