RAB31 Antibody

What is RAB31 and what cellular functions does it perform?

RAB31 (also known as RAB22B) belongs to the RAB family of small GTPases that regulate intracellular membrane trafficking. It functions as a molecular switch cycling between inactive GDP-bound and active GTP-bound forms. RAB31 is primarily localized to the trans-Golgi network (TGN) and endosomes, where it mediates several critical cellular processes:

• Transport of mannose 6-phosphate receptors (M6PRs) from the trans-Golgi network to endosomes

• Insulin-stimulated translocation of GLUT4 to the cell membrane

• Internalization of epidermal growth factor receptor (EGFR) from the cell membrane into endosomes

• Maturation of phagosomes that engulf pathogens such as S. aureus and M. tuberculosis

• Regulation of exosome biogenesis and secretion

RAB31 interacts with several effector proteins including early endosome antigen 1 (EEA1), and its activity is regulated by GTPase-activating proteins (GAPs) and guanine nucleotide exchange factors (GEFs) such as GAPex-5 and RIN3 .

What types of RAB31 antibodies are available for research?

Several types of RAB31 antibodies are commercially available for research applications:

These antibodies have been validated in multiple applications and are suitable for various experimental approaches depending on the research question .

How should I validate the specificity of a RAB31 antibody?

Validating antibody specificity is crucial for reliable experimental results. For RAB31 antibodies, consider these methodological approaches:

• Cross-reactivity testing with related Rab proteins: Test against closely related Rab family members, particularly Rab5 and Rab22A, which share high sequence homology with RAB31 (approximately 86.8% and 91.6% homology, respectively) .

• Knockdown/knockout validation: Use siRNA, shRNA, or CRISPR-Cas9 to knockdown or knockout RAB31 expression. For example, researchers have validated RAB31 antibodies using shRNA (shRNA1 and shRNA2) in SCC-4 and SCC-25 cell lines, confirming significant reduction in both protein and mRNA levels .

• One-sided ELISA: This assay can confirm specificity by comparing antibody binding to RAB31 versus control proteins. For instance, the polyclonal antibody pAb RT3-IgG showed strong reaction with recombinant RAB31-His and GST-RAB31 but no reaction with BSA .

• Western blot analysis: Test antibody specificity using cell lines known to express RAB31 at different levels. The CAL-27, SCC-4, and SCC-25 oral squamous cell carcinoma cell lines have been documented to express RAB31, while SCC-9 cells show lower expression levels comparable to normal controls .

• Multiple antibody comparison: Use different antibodies targeting distinct epitopes of RAB31 to confirm consistent detection patterns .

What are the optimal conditions for using RAB31 antibodies in Western blotting?

Based on published protocols, these are the recommended conditions for Western blotting with RAB31 antibodies:

For example, Western blot analysis of RAB31 in various cell lines using antibody at 1:1000 dilution successfully detected the 22 kDa protein in multiple samples . When examining RAB31 overexpression, a significant increase in band intensity at 22 kDa can be observed compared to control transfected cells .

How can I optimize immunohistochemistry protocols for RAB31 antibodies?

For effective immunohistochemical (IHC) detection of RAB31, consider these methodological recommendations:

• Tissue preparation: Use formalin-fixed, paraffin-embedded (FFPE) tissue sections. RAB31 has been successfully detected in various tissues including colon, breast, and oral mucosa .

• Antigen retrieval: Use either TE buffer pH 9.0 or citrate buffer pH 6.0. For optimal results, perform microwave-based antigen retrieval .

• Blocking: Incubate sections with an endogenous peroxidase blocker for 15 minutes prior to primary antibody application .

• Primary antibody incubation: Use RAB31 antibody at 1:50-1:500 dilution (optimize for each antibody) and incubate overnight at 4°C .

• Secondary antibody: Incubate with appropriate HRP-conjugated secondary antibody for 1 hour at room temperature .

• Visualization: Use DAB (3,3'-diaminobenzidine) for chromogenic detection followed by hematoxylin counterstaining .

• Scoring system: Consider using a semi-quantitative scoring system based on staining intensity (0-3) and percentage of RAB31-positive cells (0-3) as described in gastric cancer studies .

When optimized, RAB31 typically shows cytoplasmic staining with occasional perinuclear and/or nuclear staining in cancer cells, with stromal cells showing less frequent staining .

How can RAB31 antibodies be used to investigate its role in cancer progression?

RAB31 has been implicated in various cancers, and antibodies against RAB31 can be valuable tools to investigate its role in cancer progression:

• Expression level analysis: Use RAB31 antibodies for Western blot, IHC, or ELISA to compare expression levels between normal tissues and cancer samples. Studies have shown that RAB31 expression correlates with cancer progression and patient prognosis in several cancer types .

• Correlation with clinical parameters: In oral squamous cell carcinoma (OSCC), RAB31 positivity correlates with pathological grade and patient survival. Below is a representative data table from OSCC research:

• Functional studies using knockdown/overexpression models: After modifying RAB31 expression through knockdown or overexpression, use RAB31 antibodies to:

  • Confirm successful manipulation of protein levels

  • Track changes in cellular localization

  • Monitor effects on downstream signaling pathways

  For example, in SCC-4 and SCC-25 cell lines, RAB31 knockdown led to decreased expression of survivin, cyclin D1, N-cadherin, and MMP-9, with increased expression of E-cadherin and BCL2 .

• EMT marker correlation: Use RAB31 antibodies alongside epithelial-mesenchymal transition (EMT) markers to investigate correlations. Studies in colorectal cancer have shown that RAB31 overexpression suppresses epithelial marker CDH1 while increasing mesenchymal markers SNAI1 and SNAI2 .

• Exosome analysis: RAB31 antibodies can be used to study its role in exosome secretion, which may contribute to metastasis. In gastric cancer, RAB31 overexpression enhanced exosome secretion, and these exosomes promoted pulmonary metastasis in vivo .

How can I use RAB31 antibodies to study vesicular trafficking dynamics?

RAB31 plays critical roles in vesicular trafficking, and antibodies can be powerful tools to investigate these processes:

• Co-localization studies: Use immunofluorescence with RAB31 antibodies alongside markers for different cellular compartments to track protein trafficking:

  • Trans-Golgi network: TGN46, Golgin-97

  • Early endosomes: EEA1, Rab5

  • Late endosomes: Rab7, CD63

  • Recycling endosomes: Rab11

• Tracking receptor internalization: RAB31 is involved in EGFR internalization. Design pulse-chase experiments where cells are stimulated with EGF, then fixed at different time points and stained for both RAB31 and EGFR to track co-localization patterns .

• Live-cell imaging: Combine RAB31 antibodies with fluorescently-tagged cargo proteins to monitor trafficking in real-time using techniques like FRAP (Fluorescence Recovery After Photobleaching) or FLIP (Fluorescence Loss In Photobleaching).

• Exosome biogenesis: RAB31 marks and controls an ESCRT-independent exosome pathway. Use RAB31 antibodies to investigate how it interacts with flotillin proteins in lipid raft microdomains to drive EGFR entry into multivesicular endosomes (MVEs) .

• Immunoprecipitation for interaction studies: Use RAB31 antibodies for co-immunoprecipitation to identify interacting partners involved in trafficking. For example, RAB31/AGR2 axis has been shown to mediate exocytosis in colorectal cancer cells .

How can I investigate RAB31's role in therapeutic resistance mechanisms?

RAB31 has been implicated in therapeutic resistance, particularly in cancer. Here's how to use RAB31 antibodies to study these mechanisms:

• Expression analysis in resistant cell lines: Compare RAB31 protein levels between parental and drug-resistant cancer cell lines using Western blot or immunofluorescence. Studies have shown significant increases in RAB31 expression in oxaliplatin-resistant colorectal cancer cells compared to parental or other chemotherapy-resistant groups .

• Correlation with resistance markers: Use RAB31 antibodies alongside other markers of resistance (e.g., drug efflux transporters, anti-apoptotic proteins) to establish correlations.

• Extracellular vesicle characterization: Since RAB31 regulates exosome secretion, use antibodies to study how RAB31-dependent vesicles might contribute to drug resistance:

  • Isolate exosomes from RAB31-overexpressing and control cells

  • Use RAB31 antibodies together with exosome markers (CD63, CD81, TSG101) to characterize vesicle content

  • Track transfer of resistance through exosomes

• Signaling pathway analysis: After drug treatment, use RAB31 antibodies in combination with phospho-specific antibodies to study alterations in signaling pathways that might contribute to resistance.

• In vivo resistance models: In xenograft models, use RAB31 antibodies for IHC to correlate tumor RAB31 expression with response to therapy .

How do I troubleshoot non-specific binding of RAB31 antibodies?

Non-specific binding can compromise experimental results. Here are methodological approaches to troubleshoot RAB31 antibody specificity issues:

• Blocking peptide competition: Use a synthetic peptide containing the epitope recognized by the RAB31 antibody to block specific binding sites. Compare staining patterns between blocked and unblocked antibody to identify non-specific signals .

• Gradient antibody titration: Test a range of antibody dilutions (e.g., 1:100, 1:500, 1:1000, 1:2000) to determine the optimal concentration that maximizes specific signal while minimizing background .

• Alternative blocking agents: If using milk for blocking, try BSA instead, or vice versa. Different blocking agents can differentially affect non-specific binding patterns .

• Cross-adsorption: Pre-incubate the antibody with lysates from cells that don't express RAB31 to adsorb antibodies that bind to off-target proteins.

• Knockout/knockdown controls: Always include RAB31 knockout or knockdown samples as negative controls to identify non-specific bands or staining patterns .

• Alternative detection methods: If experiencing high background in chromogenic IHC, switch to fluorescence-based detection which may offer better signal-to-noise ratio.

• RAB family cross-reactivity: Due to high homology with other Rab proteins (particularly Rab5 and Rab22A), confirm specificity using recombinant proteins of these family members as described in studies with pAb RT3-IgG .

What could explain discrepancies in RAB31 detection between different antibodies?

Researchers may encounter discrepancies when using different RAB31 antibodies. Understanding these variations is important for accurate data interpretation:

• Epitope differences: Antibodies targeting different regions of RAB31 may yield different results based on:

  • Conformational changes in the protein

  • Post-translational modifications masking certain epitopes

  • Protein-protein interactions obscuring specific regions

• Active vs. inactive conformations: RAB31 cycles between GTP-bound (active) and GDP-bound (inactive) states. Some antibodies may preferentially recognize one conformation over the other.

• Splice variants: Although not extensively documented for RAB31, potential splice variants could be differentially detected by antibodies targeting different regions.

• Fixation sensitivity: For IHC applications, some epitopes may be more sensitive to formalin fixation than others. Consider comparing multiple fixation methods if discrepancies arise.

• Application-specific optimization: An antibody that works well for Western blot may not be optimal for IHC or IP. Each application may require a different antibody or optimization protocol.

• Cross-reactivity profiles: Different antibodies have different cross-reactivity patterns with related Rab proteins. The pAb RT3-IgG shows no cross-reactivity with Rab5 and minimal cross-reactivity with Rab22A despite high sequence homology .

How are RAB31 antibodies being used to investigate its role in diabetes and metabolic disorders?

Recent research has begun to elucidate RAB31's role in diabetes and metabolic disorders, with antibodies serving as key investigative tools:

• Interaction with RAGE signaling: RAB31 has been identified as an interacting protein with the receptor for advanced glycation end products (RAGE). RAB31 antibodies can be used for co-immunoprecipitation and immunostaining assays to verify this interaction, which is enhanced by glycation-serum stimulation .

• Protection against β-cell apoptosis: Studies have shown that RAB31 promotes RAGE endocytosis and inhibits glycation-serum-induced β-cell apoptosis through the pAKT/BCL2 pathway. RAB31 antibodies can be used to:

  • Track changes in RAB31 expression in response to diabetic conditions

  • Monitor its subcellular localization during RAGE signaling

  • Investigate its protective role in β-cell survival

• GLUT4 trafficking: RAB31 plays a role in insulin-stimulated translocation of GLUT4 to the cell membrane. Researchers can use antibodies to study how RAB31 expression and localization change in response to insulin and how these changes might be disrupted in insulin resistance .

• Potential therapeutic target: As RAB31 has been identified as a "promising therapeutic target for preserving functional β cells under diabetes conditions," antibodies can help validate its expression and function in preclinical models .

How can RAB31 antibodies help investigate its role in epithelial-mesenchymal transition (EMT)?

EMT is a critical process in development and disease, particularly cancer progression. RAB31 antibodies can be valuable tools to study its role in EMT:

• Correlation with EMT markers: Use RAB31 antibodies alongside established EMT markers in cancer tissues and cell lines:

  • Epithelial markers: E-cadherin, claudins, cytokeratins

  • Mesenchymal markers: N-cadherin, vimentin, SNAI1, SNAI2

  Studies in colorectal cancer have shown that RAB31 expression inversely correlates with epithelial marker CDH1 and positively correlates with mesenchymal markers SNAI1 and SNAI2 .

• TGF-β signaling modulation: Research has identified RAB31-dependent regulation of TGF-β expression and signaling in breast cancer cells. RAB31 overexpression led to strong down-regulation of TGFB1 mRNA levels in breast cancer cell lines, with concomitant reduction in both intracellular and secreted TGF-β1 protein levels .

• RAB31 modulation effects on EMT: Compare EMT marker expression before and after RAB31 knockdown or overexpression:

  • In oral squamous cell carcinoma, RAB31 silencing increased E-cadherin expression while decreasing N-cadherin and MMP-9 levels

  • In colorectal cancer, RAB31 overexpression promoted EMT and was associated with oxaliplatin resistance

• Extracellular vesicle-mediated EMT: Since RAB31 regulates exosome biogenesis and secretion, investigate how RAB31-dependent vesicles might transfer EMT-promoting factors to recipient cells using co-culture experiments and antibody-based detection methods .

How can RAB31 antibodies be used to understand its role in neurological disorders?

While less extensively studied in neurological contexts, RAB31's enrichment in brain tissues suggests potential importance:

• Expression analysis in neural tissues: Use RAB31 antibodies for IHC or Western blot to map its expression pattern in different brain regions and cell types. Studies have shown enrichment of RAB31 protein in brain tissues .

• Trafficking in neurons: Investigate RAB31's role in neuronal vesicle trafficking using co-localization studies with synaptic vesicle markers or neurotransmitter receptors.

• Glioblastoma research: RAB31 has been identified as an influential gene in glioblastoma development and is associated with survival outcomes. Antibodies can help characterize its expression and function in this context .

• Correlation with neurological disease markers: Use RAB31 antibodies alongside markers for neurodegeneration, inflammation, or other pathological processes to identify potential associations.