RAB3C Antibody

What is RAB3C and what are its primary cellular functions?

RAB3C is a small GTP-binding protein of the Ras superfamily, specifically belonging to the RAB3 family which includes four highly homologous proteins: RAB3A, RAB3B, RAB3C, and RAB3D. RAB3C functions as a peripheral membrane protein involved in:

• Membrane trafficking and vesicle formation

• Exocytosis regulation

• Synaptic vesicle cycling in neurons

RAB3C is localized on synaptic vesicles in neurons and undergoes membrane dissociation-association cycles during synaptic vesicle recycling. During neurotransmitter release, RAB3C dissociates from synaptic vesicle membranes, paralleling the behavior of RAB3A under the same conditions .

RAB3C, like other RAB proteins, cycles between a GDP-bound inactive state and a GTP-bound vesicle-associated active state. This cycling is crucial for its function in regulating vesicle transport, docking, fusion, and calcium-dependent neurotransmitter release .

How does RAB3C expression differ across tissues?

RAB3C shows a tissue-specific expression pattern:

Unlike RAB3B and RAB3D, which are predominantly expressed in non-neuronal tissues such as the parotid gland, pancreas, and adipose tissue, RAB3C and RAB3A are primarily found in neuronal and neuroendocrine cells .

What are the recommended applications for RAB3C antibodies in research?

Based on validated antibody products, RAB3C antibodies can be used in multiple experimental applications:

When designing experiments, it's crucial to optimize conditions for each specific antibody and experimental system.

How can researchers investigate RAB3C's role in synaptic vesicle dynamics?

To study RAB3C's role in synaptic vesicle dynamics, researchers should consider these methodological approaches:

• Subcellular fractionation and co-purification: RAB3C copurifies with RAB3A during synaptic vesicle isolation. Use differential centrifugation to isolate synaptic vesicles, followed by immunoblotting to detect RAB3C .

• Organelle immunoisolation: Use monoclonal antibodies directed against RAB3A to isolate vesicles, then assess RAB3C co-enrichment to demonstrate colocalization on the same organelles .

• Stimulation-induced dissociation assays: In isolated nerve terminals, stimulate neurotransmitter release and measure RAB3C dissociation from synaptic vesicle membranes. Compare with RAB3A dissociation and use RAB5 (localized on early endosomes) as a negative control since it doesn't show membrane dissociation during exocytosis .

• Immunofluorescence colocalization: Perform double-labeling experiments with RAB3C antibodies and established synaptic vesicle markers. For primary neurons, fixation in 4% paraformaldehyde (15 min at room temperature) has been validated for RAB3C detection .

• Genetic manipulation models: Study effects of RAB3C knockdown or overexpression on vesicle dynamics using time-lapse imaging and specific vesicle markers.

What is known about RAB3C's role in cancer progression and drug resistance?

RAB3C has emerged as a significant factor in colorectal cancer (CRC) progression through several mechanisms:

• Exocytosis promotion: RAB3C overexpression enhances exocytosis in CRC cells, which contributes to:

  • Cytokine/chemokine secretion

  • Drug efflux

  • Autocrine pathway activation

• Drug resistance: RAB3C overexpression increases resistance to several standard chemotherapeutic drugs:

  • 5-FU

  • Oxaliplatin

  • Regorafenib

• Molecular mechanisms:

  • RAB3C induces dystrophin expression which promotes vesicle formation and packaging

  • The RAB3C-dystrophin axis is positively correlated with PIK3CA genetic alterations in CRC patients

  • RAB3C promotes cancer metastasis through the IL-6/STAT3 axis

• Prognostic significance: Combined expression profiles of RAB3C and dystrophin serve as an independent prognostic factor in CRC and are associated with several clinicopathological parameters .

For investigating RAB3C in cancer contexts, researchers should consider:

• Creating RAB3C-overexpression models in appropriate cell lines

• Using proteomic approaches to identify RAB3C-dependent changes

• Employing tissue microarrays (TMAs) for immunohistochemical analysis of patient samples

• Developing four-point staining-intensity scoring systems (as detailed in )

What methodological approaches are recommended for visualizing RAB3C in different subcellular compartments?

For optimal subcellular visualization of RAB3C, researchers should consider these validated methodological approaches:

• Neuronal cells immunofluorescence:

  • Fix DIV9 rat E18 primary hippocampal neurons in 4% paraformaldehyde (RT, 15 min)

  • Use RAB3C antibody at 1:500 dilution

  • Co-stain with neuronal markers like β-Tubulin 3/Tuj1 (1:500) to visualize neuronal processes

  • Counterstain nuclei with DAPI

• Non-neuronal cells:

  • For HepG2 cells, use RAB3C-specific antibody at 1:25 dilution

  • Visualize with fluorophore-conjugated secondary antibodies (e.g., Rhodamine-Goat anti-Rabbit IgG)

• Tissue immunohistochemistry:

  • For formalin-fixed paraffin-embedded sections, use antigen retrieval with citrate buffer (pH 6.0) or TE buffer (pH 9.0)

  • RAB3C antibody dilution: 1:50-1:500

  • Detect using appropriate visualization systems (e.g., EnVision dual-link-system HRP detection kit)

• Scoring systems for expression analysis:

  • Use a four-point staining-intensity scoring system (0-3)

  • Calculate final IHC scores (0-300) by multiplying staining intensity score by percentage of positive cells

  • Stratify cases using appropriate cutoffs (e.g., 50%)

Unlike integral membrane proteins of synaptic vesicles, RAB3C is absent from the Golgi complex, which prevents immunostaining of the axo-dendritic region that can occur with proteins like synaptophysin, synaptobrevin/VAMP, or synaptogyrin .

How can researchers distinguish between RAB3C and other highly homologous RAB3 family members?

Distinguishing between the highly homologous RAB3 family members (RAB3A, RAB3B, RAB3C, and RAB3D) requires careful methodological approaches:

• Antibody selection and validation:

  • Use RAB3C-specific antibodies that target unique epitopes or C-terminal regions

  • Validate antibody specificity using knockout/knockdown controls

  • Confirm single band detection at the expected molecular weight (26-28 kDa)

• Expression pattern analysis:

  • RAB3A and RAB3C: primarily neuronal/neuroendocrine cells

  • RAB3B and RAB3D: predominantly non-neuronal tissues (parotid gland, pancreas, mast cells, adipose tissue)

• Functional differentiation experiments:

  • Design siRNA knockdowns specific to each RAB3 isoform

  • Perform rescue experiments with isoform-specific constructs

  • Analyze phenotypic differences in vesicle trafficking, exocytosis, or drug response

• Protein interaction studies:

  • Investigate isoform-specific binding partners using immunoprecipitation

  • In RAB3C studies, particularly examine interactions with dystrophin

• Molecular techniques:

  • Use RT-qPCR with isoform-specific primers to quantify mRNA expression

  • Employ proteomics approaches to identify isoform-specific post-translational modifications

What are the optimal protocols for Western blot detection of RAB3C?

For optimal Western blot detection of RAB3C, researchers should follow these validated protocols:

• Sample preparation:

  • For brain tissue: Standard protein extraction methods yield detectable RAB3C

  • For other tissues: Enrichment of membrane fractions may improve detection

  • Recommended protein load: 50 μg per lane for tissue extracts

• Gel electrophoresis:

  • 12% SDS-PAGE provides optimal resolution for RAB3C (26-28 kDa)

• Antibody conditions:

  • Primary antibody dilution range: 1:500-1:10000 (antibody-dependent)

  • Validated example: RAB3C antibody [16867-1-AP] at 1:300 dilution, room temperature, 1.5 hours

  • Secondary antibody: HRP-conjugated anti-rabbit IgG

• Expected results:

  • RAB3C typically appears as a single band at 26-28 kDa

  • Human brain tissue consistently shows strong expression

• Controls:

  • Positive control: Human or mouse brain tissue lysate

  • Negative control: Tissues with low RAB3C expression or RAB3C knockout samples

What are the best methods for troubleshooting weak signals or non-specific binding with RAB3C antibodies?

When facing challenges with RAB3C antibody signals, consider these methodological solutions:

• For weak signals:

  • Increase protein loading (up to 50-100 μg per lane for Western blot)

  • Optimize antibody concentration through titration experiments

  • Extend primary antibody incubation time (overnight at 4°C)

  • Use signal enhancement systems (e.g., biotin-streptavidin amplification)

  • Verify sample integrity and RAB3C expression in the specific tissue/cell type

• For non-specific binding:

  • Increase blocking stringency (5% BSA or milk, longer blocking times)

  • Use validated antibodies with confirmed specificity (e.g., [10788-1-AP] or [16867-1-AP])

  • Optimize antibody dilution (start with manufacturer recommendations, then adjust)

  • Include additional washing steps with increased detergent concentration

  • Consider using monoclonal antibodies for higher specificity

• For immunohistochemistry/immunofluorescence optimization:

  • Test different fixation protocols (4% PFA shows good results for RAB3C)

  • Optimize antigen retrieval methods:

    • Citrate buffer (pH 6.0) or TE buffer (pH 9.0) for FFPE sections

  • Adjust antibody incubation conditions (temperature, time, concentration)

  • Use detection systems appropriate for the expected expression level

• For reproducibility concerns:

  • Standardize protocols with detailed SOPs

  • Document lot numbers and antibody sources

  • Include proper positive and negative controls in each experiment

How can researchers effectively investigate RAB3C-protein interactions in research models?

To study RAB3C-protein interactions, researchers should employ these methodological approaches:

• Co-immunoprecipitation:

  • Use 0.5-4.0 μg RAB3C antibody per 1-3 mg of total protein lysate

  • Include controls: IgG control, reverse IP with suspected interacting proteins

  • For brain tissue, validated results have been obtained with mouse brain samples

• Proximity ligation assay (PLA):

  • For detecting in situ protein-protein interactions

  • Particularly useful for studying RAB3C interactions with dystrophin

• Proteomic approaches:

  • Establish RAB3C-based proteomic datasets using RAB3C overexpression models

  • Example: RAB3C overexpression led to identification of dystrophin upregulation

• Functional validation of interactions:

  • Assess direct binding through protein-protein interaction assays

  • Evaluate functional consequences using drug resistance assays

  • Example: RAB3C-dystrophin interaction was shown to regulate drug resistance in CRC cells

• Visualization of co-localization:

  • For neuronal studies: Co-stain for RAB3C and other synaptic vesicle proteins

  • Document co-enrichment of RAB3C with other proteins (e.g., RAB3A) on the same organelles

  • Use high-resolution microscopy techniques like STED or STORM for detailed co-localization

What controls should be included in RAB3C immunohistochemistry studies for research publication?

For publication-quality RAB3C immunohistochemistry studies, include these critical controls:

• Antibody specificity controls:

  • Tissues known to be positive for RAB3C (e.g., brain tissue)

  • Negative control tissues with minimal RAB3C expression

  • Peptide competition assays to confirm antibody specificity

  • Ideally, RAB3C knockout samples when available

• Technical controls:

  • Omission of primary antibody (secondary antibody only)

  • Isotype control antibody at the same concentration

  • Serial dilutions of primary antibody to demonstrate dose-dependency

  • Batch controls across multiple staining runs for consistency

• Biological controls and validation:

  • Multiple samples (n ≥ 3) to account for biological variability

  • Correlation with mRNA expression data when possible

  • Alternative detection methods (e.g., IF, WB) to confirm findings

• Scoring and quantification:

  • Implement standardized scoring systems such as:

    • Four-point staining-intensity scoring (0-3 scale)

    • Percentage of positive cells (0-100%)

    • Final IHC scores (0-300) calculated by multiplying intensity score by percentage of positive cells

  • Blind scoring by multiple trained observers

  • Include representative images of each scoring category

• Cancer tissue studies specifics:

  • Include both tumor tissues and corresponding adjacent non-cancerous tissues

  • For tissue microarrays, select multiple cores (e.g., three 1-mm cores) from different areas of the tumor tissue

How can RAB3C antibodies be used to investigate synapse-specific defects in neurological disorders?

For investigating synapse-specific defects using RAB3C antibodies:

• Experimental design approaches:

  • Compare RAB3C localization patterns between control and disorder models

  • Quantify RAB3C-positive puncta density and size in specific brain regions

  • Assess RAB3C dissociation from synaptic vesicles during stimulation experiments

• Methodological considerations:

  • For mouse models: Brain sections can be analyzed using IHC with 1:50-1:500 antibody dilution

  • Tissue preparation: Antigen retrieval with TE buffer (pH 9.0) or citrate buffer (pH 6.0)

  • Neuronal cultures: Primary hippocampal neurons show clear RAB3C staining at DIV9

• Functional correlation:

  • Combine RAB3C immunostaining with electrophysiology recordings

  • Perform live-cell imaging with fluorescently tagged RAB3C to track dynamics

  • Compare RAB3C distribution with active zone proteins like Bruchpilot

• Insights from Drosophila research:

  • In Drosophila rab3 mutants, active zone proteins (Bruchpilot, calcium channels, T-bars) concentrate at a fraction of sites while others remain devoid of these components

  • Late addition of Rab3 rapidly reverses this phenotype by recruiting proteins to previously empty sites

  • This demonstrates Rab3's role in dynamically controlling presynaptic release machinery composition

This research direction is particularly relevant as RAB3C functions in vesicle trafficking and exocytosis, processes fundamental to synaptic transmission that are often impaired in neurological disorders.

What are the latest methodological approaches for using RAB3C antibodies in cancer research?

Recent methodological advances for RAB3C investigation in cancer research include:

• Tissue microarray (TMA) analysis:

  • Assemble TMAs containing tumor tissues and corresponding adjacent non-cancerous tissues

  • For each case, select multiple cores (three 1-mm cores) from different tumor regions

  • Use validated antibodies: anti-human RAB3C (1:100; Cat # 15029-1-AP) and dystrophin (1:50; Cat # HPA023885)

  • Implement standardized scoring systems with 0-3 intensity scale

• Combined biomarker evaluation:

  • Assess RAB3C in conjunction with dystrophin expression

  • Use the combined expression profile as a prognostic indicator

  • Correlate with clinicopathological parameters and genetic alterations (PIK3CA/KRAS)

• Drug resistance mechanisms:

  • Establish RAB3C overexpression models in cancer cell lines

  • Evaluate resistance to chemotherapeutic agents (5-FU, oxaliplatin, regorafenib)

  • Investigate reversibility of resistance through pharmacological approaches

• Potential therapeutic targets:

  • Connectivity mapping identified cannabinoid receptor 2 (CB2) agonists as compounds that may reverse RAB3C-associated drug resistance

  • These agonists showed synergistic effects when combined with standard chemotherapy regimens

  • Direct targeting of the RAB3C-dystrophin interaction represents a novel therapeutic approach

• Multi-omics integration:

  • Combine RAB3C-based transcriptomic and proteomic datasets

  • Analyze correlation with genetic alterations (PIK3CA, KRAS)

  • Identify downstream signaling pathways (e.g., IL-6/STAT3 axis)

How can researchers effectively distinguish between membrane-bound and cytosolic RAB3C in experimental models?

To differentiate between membrane-bound and cytosolic RAB3C:

• Subcellular fractionation protocol:

  • Prepare homogenates in isotonic buffer

  • Separate membrane and cytosolic fractions through differential centrifugation

  • Ultracentrifugation at 100,000g separates vesicle/membrane-bound (pellet) from soluble (supernatant) RAB3C

  • Verify fraction purity using markers for cytosol (e.g., GAPDH) and membranes (e.g., Na+/K+-ATPase)

• Biochemical approaches:

  • Treat samples with detergents that selectively solubilize membranes

  • Use carbonate extraction (pH 11.5) to distinguish peripheral (like RAB3C) from integral membrane proteins

  • Analyze GTP/GDP-bound states, as GTP-bound RAB3C predominantly associates with membranes

• Imaging strategies:

  • Immunofluorescence with minimal permeabilization to visualize membrane-associated RAB3C

  • Sequential extraction with increasing detergent concentrations before fixation

  • Super-resolution microscopy to visualize RAB3C on individual vesicles

• Dynamic studies:

  • In neuronal preparations, stimulate exocytosis to observe RAB3C dissociation from membranes

  • This parallels RAB3A behavior but differs from RAB5, which remains membrane-associated during exocytosis

  • Time-lapse imaging with fluorescently tagged RAB3C can track the cycling between membrane and cytosol

• Regulatory mechanisms:

  • Investigate GEFs (guanine nucleotide exchange factors) and GAPs (GTPase-activating proteins) that regulate RAB3C cycling

  • Study the effect of mutations that lock RAB3C in GTP-bound (constitutively active) or GDP-bound (inactive) states