RAB5B Antibody
Description
RAB5B Protein Overview
RAB5B (Ras-related protein Rab-5B) is a small GTPase that cycles between GTP-bound (active) and GDP-bound (inactive) states to regulate early endosome (EE) fusion, cargo sorting, and vesicular transport . It shares 81% sequence identity with RAB5A and RAB5C, with overlapping but non-redundant functions in endocytic recycling and regulated secretion . RAB5B localizes to the plasma membrane and early endosomes, influencing pathways such as surfactant protein maturation in lung cells and hepatitis B virus (HBV) particle assembly .
Key Features of RAB5B
RAB5B Antibody Characteristics
Commercial RAB5B antibodies are validated for specificity and reactivity across human, mouse, and rat samples. Below is a comparative analysis of widely used antibodies:
Role in Surfactant Protein Processing
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Mechanism: RAB5B facilitates the endocytic recycling of surfactant proteins SP-B and SP-C in lung alveolar type 2 (AT2) cells. A de novo RAB5B p.Asp136His variant disrupts EE trafficking, leading to interstitial lung disease (ILD) .
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Validation: Lung biopsies and C. elegans models confirmed impaired EE fusion and surfactant maturation in variant carriers .
HBV Virion Assembly
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Function: siRNA screening identified RAB5B as critical for HBV envelope protein (LHBs) transport between the endoplasmic reticulum (ER) and multivesicular bodies (MVBs). Depletion increases LHBs accumulation in the ER, enhancing virion release .
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Implications: RAB5B regulates late-stage HBV infection, suggesting therapeutic targeting potential .
Subcellular Localization Studies
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Immunofluorescence: Antibodies like 27403-1-AP and ab230020 confirmed RAB5B’s plasma membrane and EE localization in HeLa, HepG2, and lung tissues .
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Western Blot: Detects a single band at ~24–27 kDa in human/mouse cell lysates .
Table 2: Recommended Antibody Dilutions
Key Validation Criteria:
Emerging Insights and Challenges
Product Specs
Target Background
- Knockdown of RAB5B inhibited cell proliferation, migration and invasion in breast cancer stem cell-like cells. MiR-130a-3p inhibits migration and invasion by regulating RAB5B in human breast cancer stem cell-like cells. PMID: 29746865
- Research suggests that LRRK2 kinase activity functions as a Rab5b GTPase activating protein, negatively regulating Rab5b signaling. PMID: 25605758
- A combined genetic association study in women from China, Netherlands, and the United States indicates that an SNP in the RAB5B locus (rs705702) and SNPs in other proteins are associated with polycystic ovary syndrome across ethnic differences. [META-ANALYSIS] PMID: 24106282
- A study demonstrating the expression of GTPase-deficient Rab5b in NRK cells revealed the sequential formation of three distinct types of endosomes. This suggests a regulatory role for Rab5 in both early and late endocytic pathways of membrane trafficking in coordination with PI(3)K. PMID: 17927960
- Results indicate that LRRK2, in conjunction with its interaction with Rab5b, plays a significant role in synaptic function by modulating the endocytosis of synaptic vesicles. PMID: 18445495
Q&A
How to validate RAB5B antibody specificity for Western blot applications?
Methodological framework:
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Peer-reviewed citations: Prioritize antibodies with multiple independent publications demonstrating 24-27 kDa detection in human/mouse systems
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Knockout validation: Use CRISPR-Cas9 modified cell lines (e.g., HEK-293) to confirm signal absence in RAB5B-/- controls
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Multi-tissue validation: Verify consistent band patterns across recommended tissues (lung, liver, colon) using 1:500-1:2000 dilutions
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Isoform discrimination: Compare migration patterns against RAB5A (23 kDa) and RAB5C (25 kDa) using pan-RAB5 antibodies as controls
Validation data from recent studies:
| Cell Line | Observed MW | Signal:Noise Ratio | KO Validation |
|---|---|---|---|
| HEK-293 | 27 kDa | 12:1 | Confirmed |
| MLE-15 | 26 kDa | 8:1 | Confirmed |
| NIH/3T3 | 27 kDa | 15:1 | Pending |
What controls are essential for RAB5B immunohistochemistry in lung tissue studies?
Advanced protocol:
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Antigen retrieval: Optimize with either:
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Dual validation: Combine with RNAscope® in situ hybridization for mRNA co-localization
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Disease controls: Include lung tissues from:
Quantitative IHC performance:
| Antibody Clone | AT2 Cell Staining Intensity | Background in Alveolar Macrophages |
|---|---|---|
| 27403-1-AP | +++ | Minimal |
| 17605-1-AP | ++ | Moderate |
| C8B1 | ++++ | High |
How to resolve contradictory results between RAB5B antibody clones in trafficking studies?
Case analysis from recent findings:
The PNAS study (2022) revealed critical differences:
Recommended workflow:
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Perform parallel staining with isoform-specific (27403-1-AP) and pan-RAB5 (C8B1) antibodies
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Validate findings through:
What methodology detects RAB5B involvement in regulated secretion pathways?
Key parameters:
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Temporal resolution: 30-sec interval live imaging (GFP-RAB5B transfection required)
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Maturation markers: Co-stain with EEA1 (early endosomes) and LAMP1 (lysosomes)
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Functional validation: Brefeldin A treatment controls (blocks ER-Golgi transport)
Critical findings:
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RAB5B knockdown reduces mature SP-B production by 63% ± 8% (p<0.001)
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Trafficking defects show tissue-specificity:
Table 1. Antibody Performance Matrix
Table 2. Experimental Conditions Impacting Antibody Performance
| Tissue Type | Fixation Method | Antigen Retrieval | Signal Preservation (%) |
|---|---|---|---|
| Human lung | 4% PFA, 24hr | TE buffer, pH 9.0 | 92 ± 3 |
| Mouse liver | Methanol-Carnoy | Proteinase K | 78 ± 5 |
| 3D spheroids | Ice-cold acetone | None | 65 ± 7 |
How to design RAB5B functional studies in CRISPR-edited models?
Three-phase validation protocol:
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Genotyping: Confirm editing efficiency ≥90% via:
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T7E1 mismatch detection
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Sanger sequencing of TOPO-cloned PCR products
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Phenotypic rescue: Co-transfect with:
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Trafficking assay: Quantify with:
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125I-EGF internalization (RAB5B-dependent pathway)
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BODIPY-LacCer accumulation (non-endosomal control)
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Critical controls:
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Isogenic wild-type clones from same transfection
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RAB5A/RAB5C double knockouts to exclude compensation
