rab11fip4b Antibody

What is RAB11FIP4B and how does it differ from other RAB11FIP family members?

RAB11FIP4B is an N-terminally truncated isoform of the RAB11FIP4 protein, which functions as a regulator of endocytic trafficking by participating in membrane delivery processes. While RAB11FIP4A represents the full-length protein predominantly expressed in neural tissues, RAB11FIP4B is a shorter variant that appears to be expressed more ubiquitously, albeit at lower levels .

The RAB11FIP family consists of five proteins (FIP1-5) that interact with RAB11 GTPases to regulate vesicular trafficking. Class II FIPs, which include RAB11FIP3 and RAB11FIP4, contain EF-hand domains and lack the phospholipid-binding C2 domains found in class I FIPs . RAB11FIP4B specifically is essential for the abscission step in cytokinesis, potentially functioning as an 'address tag' that directs recycling endosome membranes to the cleavage furrow during late cytokinesis.

What validation methods should researchers employ to ensure RAB11FIP4B antibody specificity?

Researchers should implement multiple validation strategies aligned with the "five pillars" of antibody validation to ensure specificity:

• Genetic strategy: Use knockout or knockdown cell lines as negative controls. This is considered the gold standard for antibody validation. For RAB11FIP4B, CRISPR/Cas9 knockout systems have been successfully used to validate antibody specificity . Compare staining patterns between wild-type cells and those with RAB11FIP4B knocked out to confirm specificity.

• Orthogonal strategy: Compare antibody-based protein detection with antibody-independent methods such as mass spectrometry or RNA expression data. For RAB11FIP4B, a minimum of fivefold difference in RNA expression levels between samples is recommended for meaningful correlation .

• Independent antibody verification: Use two or more antibodies targeting different, non-overlapping epitopes of RAB11FIP4B. Concordant results from antibodies recognizing different regions strongly indicate specificity .

• Recombinant expression: Test the antibody in a system where RAB11FIP4B is overexpressed. The antibody should show stronger signal in cells with recombinant expression compared to control cells .

• Immunoprecipitation followed by mass spectrometry: This method can confirm that the antibody is capturing the intended target rather than cross-reacting with other proteins .

For RAB11FIP4B antibodies specifically, validation data shows that genetic approaches yield more reliable results than orthogonal methods, especially for immunofluorescence applications .

How should Western blot protocols be optimized for RAB11FIP4B detection?

Optimizing Western blot protocols for RAB11FIP4B detection requires attention to several technical aspects:

When troubleshooting Western blots with RAB11FIP4B antibodies, researchers should pay particular attention to the predicted molecular weight and be aware that post-translational modifications may affect migration patterns. Additionally, cross-reactivity with RAB11FIP4A must be considered when interpreting results, which requires careful antibody selection targeting unique regions of RAB11FIP4B .

What are the best experimental designs to study RAB11FIP4B interactions with Rab GTPases?

To effectively study RAB11FIP4B interactions with Rab GTPases, researchers should employ multi-faceted approaches:

• Co-immunoprecipitation (Co-IP): Use RAB11FIP4B antibodies to pull down protein complexes, followed by Western blotting for Rab11 or other GTPases. This technique directly demonstrates physical interaction in cell lysates. When performing Co-IP, it's crucial to use antibodies targeting regions outside the Rab11-binding domain to avoid interference with the interaction .

• Proximity Ligation Assay (PLA): This technique allows visualization of protein-protein interactions in situ with high sensitivity. PLA produces fluorescent spots only when the two target proteins are in close proximity (<40 nm), providing spatial information about interactions within cells.

• Fluorescence Resonance Energy Transfer (FRET): By tagging RAB11FIP4B and Rab GTPases with appropriate fluorophore pairs, researchers can measure energy transfer that occurs only when proteins are in very close proximity (typically <10 nm).

• Yeast Two-Hybrid or Mammalian Two-Hybrid: These systems can validate direct interactions and map interaction domains. Studies have revealed that RAB11FIP4 interacts with Rab11 through its C-terminal coiled-coil domain .

• Domain mapping experiments: Using truncation mutants to identify specific interaction regions. Research has shown that the C-terminal region containing a conserved Rab-binding domain (RBD) is required for RAB11FIP4B interaction with Rab11, while the N-terminal region containing the EF-hand motif mediates other functions .

It's important to note that RAB11FIP4 can function through both Rab11-dependent and Rab11-independent mechanisms. Studies have shown that some functions of RAB11FIP4 require the ARF6 GTPase rather than Rab11 , suggesting that experimental designs should account for multiple potential interaction partners.

How can RAB11FIP4B antibodies be optimized for immunofluorescence and immunohistochemistry applications?

Optimizing RAB11FIP4B antibodies for immunostaining requires careful attention to fixation, permeabilization, and blocking conditions:

• Fixation method selection:

  • For immunofluorescence: 4% paraformaldehyde (10-15 minutes at room temperature) preserves subcellular structure while maintaining antigenicity

  • For immunohistochemistry: 10% neutral buffered formalin is standard for FFPE tissues, though antigen retrieval becomes critical

• Permeabilization optimization:

  • 0.1-0.3% Triton X-100 for 5-10 minutes works well for accessing intracellular epitopes

  • For membrane-associated proteins like RAB11FIP4B, gentler permeabilization with 0.1% saponin may better preserve localization patterns

• Antigen retrieval for IHC:

  • Heat-induced epitope retrieval (HIER) with citrate buffer (pH 6.0) or EDTA buffer (pH 9.0)

  • For RAB11FIP4B, comparative studies suggest citrate buffer generally yields better results

• Blocking and antibody conditions:

  • 5-10% normal serum (matching species of secondary antibody) with 1% BSA

  • Primary antibody dilutions typically range from 1:100-1:500 for IF and IHC

  • Overnight incubation at 4°C generally improves specific signal-to-noise ratio

• Controls:

  • Peptide competition assays where pre-incubation with the immunizing peptide should abolish specific staining

  • Knockout/knockdown controls are especially important for validating subcellular localization patterns

For RAB11FIP4B, immunofluorescence studies have revealed localization to recycling endosome membranes, the cleavage furrow during cytokinesis, and the midbody. When interpreting staining patterns, researchers should be aware that genetic validation approaches have proven more reliable than orthogonal methods for confirming antibody specificity in immunofluorescence applications, with studies showing that 80% of antibodies validated by genetic strategies perform as expected compared to only 38% of those validated by orthogonal methods .

What are the challenges in detecting post-translational modifications of RAB11FIP4B?

Detecting post-translational modifications (PTMs) of RAB11FIP4B presents several technical challenges:

Given that RAB11FIP4B participates in dynamic cellular processes like cytokinesis and endosomal trafficking, phosphorylation is likely to be an important regulatory mechanism . Recent studies of other RAB11FIP family members suggest that phosphorylation may regulate their interactions with Rab GTPases and influence subcellular localization.

How can RAB11FIP4B antibodies help elucidate its role in disease models?

RAB11FIP4B antibodies serve as critical tools for investigating the protein's involvement in disease pathology through several methodological approaches:

• Expression level analysis in disease states:

  • Studies have shown that RAB11FIP4 is downregulated in cystinosis, a lysosomal storage disease . Using validated antibodies, researchers can quantify RAB11FIP4B levels in patient-derived samples or disease models via Western blotting or immunohistochemistry.

  • In contrast, RAB11FIP4 overexpression correlates with poor prognosis in pancreatic cancer . Immunohistochemical scoring of tumor samples using validated antibodies can help establish RAB11FIP4B as a prognostic biomarker.

• Mechanistic studies in cellular models:

  • In cystinosis models, reconstitution of RAB11FIP4 expression reduced endoplasmic reticulum stress, decreased oxidative stress, and restored normal autophagosome levels . Antibodies can track these effects by monitoring RAB11FIP4B-dependent changes in stress markers and trafficking proteins.

  • For cancer studies, antibodies can help elucidate how RAB11FIP4B affects cell proliferation, invasion, and migration by examining its interaction partners and downstream effectors.

• Subcellular localization in pathological conditions:

  • Immunofluorescence with RAB11FIP4B antibodies can reveal alterations in protein localization during disease states. For example, in cystinotic cells, RAB11FIP4 reconstitution increased LAMP2A localization at the lysosomal membrane and megalin distribution at the plasma membrane .

• Therapeutic target validation:

  • Antibodies can confirm target engagement in experimental therapies aimed at modulating RAB11FIP4B levels or function. In cystinosis models, treatment with genistein or the CMA activator QX77 restored RAB11FIP4 expression levels .

• Interaction with disease-associated proteins:

  • Co-immunoprecipitation using RAB11FIP4B antibodies can identify altered protein interactions in disease states, especially with Rab11, ARF6, and components of trafficking machinery.

The evidence suggests that RAB11FIP4B dysregulation contributes to disease through altered vesicular trafficking, highlighting its potential as both a biomarker and therapeutic target .

What methodological considerations are important when studying RAB11FIP4B in primary cells versus cell lines?

When investigating RAB11FIP4B in different cellular systems, researchers should consider these methodological differences:

When studying RAB11FIP4B's role in specific diseases like cystinosis or cancer, researchers should prioritize patient-derived primary cells while using cell lines for mechanistic studies and initial antibody validation . For developmental studies examining RAB11FIP4B's role in processes like retinal progenitor differentiation, primary cells or organoid models provide more physiologically relevant systems than immortalized lines .

How can quantitative microscopy be optimized for RAB11FIP4B localization studies?

Quantitative microscopy for RAB11FIP4B localization requires rigorous optimization of imaging parameters and analytical approaches:

• Image acquisition optimization:

  • Use high numerical aperture objectives (1.3-1.4 NA) to maximize resolution

  • Implement deconvolution or super-resolution techniques (e.g., Airyscan) for detailed subcellular localization, particularly important for distinguishing between RAB11FIP4B's multiple locations (recycling endosomes, cleavage furrow, midbody)

  • Maintain consistent exposure settings across experimental groups

  • Include reference standards in each imaging session for normalization

• Colocalization analysis protocols:

  • Employ dual or triple labeling with established markers for specific subcellular compartments:

    • Rab11 for recycling endosomes

    • LAMP2A for lysosomes

    • Megalin for plasma membrane in proximal tubule cells

  • Calculate Pearson's correlation coefficient or Manders' overlap coefficient for quantitative assessment

  • Use object-based colocalization for punctate structures

• Dynamics and trafficking analysis:

  • Implement FRAP (Fluorescence Recovery After Photobleaching) to assess mobility

  • Use live-cell imaging with photoswitchable fusion proteins to track trafficking routes

  • Consider pulse-chase approaches with internalized markers to follow endocytic pathways

• Quantification approaches:

  • For fluorescence intensity: measure integrated density rather than mean intensity

  • For subcellular distribution: use line scans or radial profile analysis from cell center

  • For endosomal localization: count RAB11FIP4B-positive puncta and measure distance from center

• Software and analysis tools:

  • Use specialized software (ImageJ/FIJI with appropriate plugins, CellProfiler, Imaris) for unbiased quantification

  • Implement batch processing for consistent analysis across multiple images

  • Consider machine learning approaches for complex pattern recognition

Research has demonstrated that RAB11FIP4B localization changes during cell cycle progression, with particular enrichment at the midbody during cytokinesis. Quantitative assessment of these dynamic changes requires careful experimental design, including synchronized cell populations and time-lapse imaging.

What is the current state of knowledge about RAB11FIP4B's functional domains and how can antibodies target them?

Current understanding of RAB11FIP4B's functional domains informs strategic antibody targeting for different experimental applications:

Research has demonstrated that the N-terminal region containing the EF-hand motif is essential for RAB11FIP4A's function in retinal development, while the C-terminal region is necessary but not sufficient for endosomal localization . This functional separation suggests that antibodies targeting different domains could be strategically employed:

• For distinguishing between RAB11FIP4A and RAB11FIP4B isoforms, antibodies targeting the N-terminal region present only in the full-length FIP4A are most appropriate.

• For general detection of RAB11FIP4 proteins regardless of isoform, antibodies targeting conserved internal regions or the C-terminal domain are recommended.

• For functional studies, researchers should be aware that antibodies binding the C-terminal Rab11-binding domain might interfere with protein interactions, potentially affecting experimental outcomes in live-cell applications.

The truncated N-terminus of RAB11FIP4B compared to RAB11FIP4A suggests potentially distinct functional properties between these isoforms, though further research is needed to fully characterize these differences .

How should researchers approach contradictory results from different RAB11FIP4B antibodies?

When confronted with contradictory results from different RAB11FIP4B antibodies, researchers should implement a systematic troubleshooting approach:

• Epitope mapping comparison:

  • Review the specific epitopes targeted by each antibody

  • Consider whether antibodies recognize different isoforms (RAB11FIP4A vs. RAB11FIP4B)

  • Determine if post-translational modifications might affect epitope accessibility

• Validation assessment:

  • Evaluate the validation methods used for each antibody

  • Prioritize results from antibodies validated by genetic approaches (knockout/knockdown) over those validated only by orthogonal methods

  • Consider the application specificity of validation (an antibody validated for Western blot may not work in IHC)

• Experimental condition analysis:

  • Assess differences in sample preparation, fixation, and detection methods

  • Consider cell type or tissue-specific factors that might affect results

  • Evaluate buffer conditions that might influence epitope accessibility

• Independent verification approaches:

  • Implement genetic strategies (siRNA, CRISPR) to confirm specificity

  • Use recombinant expression to verify detection capability

  • Consider orthogonal methods (mass spectrometry, RNA analysis) to resolve contradictions

• Systematic comparison workflow:

  • Test multiple antibodies side-by-side under identical conditions

  • Include appropriate positive and negative controls for each antibody

  • Document all variables systematically to identify potential sources of discrepancy

Research has shown that even antibodies from reputable sources can have significant variability in specificity and performance . A recent large-scale study demonstrated that for immunofluorescence applications, only 38% of antibodies validated by orthogonal methods performed as expected, compared to 80% of those validated by genetic approaches . This highlights the critical importance of appropriate validation strategies when resolving contradictory results.

What are the best approaches for multiplexed detection of RAB11FIP4B and its interaction partners?

Effective multiplexed detection of RAB11FIP4B and its interaction partners requires strategic planning and technical optimization:

• Multiplex immunofluorescence techniques:

  • Sequential immunostaining with careful antibody stripping between rounds

  • Tyramide signal amplification (TSA) allows antibodies from the same species to be used sequentially

  • Spectral unmixing for closely overlapping fluorophores

  • Consideration of primary antibody species combinations to avoid cross-reactivity

• Proximity-based interaction detection:

  • Proximity Ligation Assay (PLA) for detecting RAB11FIP4B interactions with Rab11, ARF6, or other partners within 40nm proximity

  • FRET/FLIM analysis for direct protein-protein interactions

  • BiFC (Bimolecular Fluorescence Complementation) for visualizing direct interactions in live cells

• Mass spectrometry-based approaches:

  • SWATH-MS or TMT labeling for quantitative comparison of interaction partners

  • IP-MS (immunoprecipitation followed by mass spectrometry) to identify the complete RAB11FIP4B interactome

  • Crosslinking mass spectrometry to map interaction interfaces

• Imaging technologies for multiplexed detection:

  • CODEX or Imaging Mass Cytometry for highly multiplexed tissue imaging

  • Multi-round immunofluorescence with photobleaching or antibody stripping

  • Super-resolution microscopy for detailed colocalization analysis

• Validation strategies for interaction partners:

  • Co-immunoprecipitation with reverse pull-down (i.e., IP with partner antibody, detect RAB11FIP4B)

  • GST pull-down assays with recombinant proteins

  • Yeast two-hybrid or mammalian two-hybrid assays for direct interactions

Research has identified key interaction partners for RAB11FIP4B, including Rab11, which binds to the C-terminal domain, and ARF6, which is involved in RAB11FIP4B's function in cytokinesis . When designing multiplexed detection strategies, researchers should consider the subcellular localization patterns of these interaction partners, as RAB11FIP4B localizes to recycling endosome membranes, the cleavage furrow during cell division, the midbody, and cytoplasmic vesicles.

How can researchers differentiate between direct and indirect effects when studying RAB11FIP4B function?

Distinguishing direct from indirect effects in RAB11FIP4B functional studies requires complementary approaches:

• Temporal analysis strategies:

  • Implement acute inactivation systems (e.g., auxin-inducible degron) to observe immediate consequences of RAB11FIP4B removal

  • Use time-course experiments after RAB11FIP4B manipulation to distinguish primary (rapid) from secondary (delayed) effects

  • Employ pulse-chase protocols to track cargo trafficking with precise temporal resolution

• Domain-specific mutant approaches:

  • Create point mutations in specific functional domains rather than complete knockout

  • Use structure-guided mutations that disrupt individual interactions (e.g., Rab11-binding domain mutants)

  • Express truncation mutants that lack specific domains, similar to studies showing the essential role of the N-terminal EF-hand domain in retinal development

• Rescue experiment design:

  • Perform phenotypic rescue with wild-type RAB11FIP4B after knockdown/knockout

  • Compare rescue efficiency of different RAB11FIP4B mutants to identify essential domains

  • Use RAB11FIP4A vs. RAB11FIP4B in rescue experiments to identify isoform-specific functions

• Interaction partner manipulation:

  • Perform simultaneous manipulation of RAB11FIP4B and its interaction partners

  • Use dominant-negative Rab11 to block Rab11-dependent functions, as demonstrated in studies showing that RAB11FIP4 operates through both Rab11-dependent and -independent mechanisms

  • Employ ARF6 inhibitors like NAV2729 to distinguish between Rab11 and ARF6-mediated functions

• Systems biology approaches:

  • Conduct network analysis of proteomic or transcriptomic changes after RAB11FIP4B manipulation

  • Use computational modeling to predict direct vs. cascade effects

  • Perform correlation analysis between RAB11FIP4B levels and cellular phenotypes across multiple conditions

Studies have demonstrated that reconstitution of RAB11FIP4 expression in cystinotic cells improves cellular homeostasis through multiple mechanisms, including decreased ER stress, reduced oxidative stress, and normalization of autophagosome levels . The observation that dominant-negative Rab11 only partially blocks these effects indicates that RAB11FIP4B functions through both Rab11-dependent and Rab11-independent pathways, highlighting the importance of mechanistic dissection in functional studies.

What considerations are important when designing knockdown/knockout experiments to validate RAB11FIP4B antibodies?

Designing effective knockdown/knockout experiments for RAB11FIP4B antibody validation requires careful planning and appropriate controls:

• Knockdown/knockout strategy selection:

  • CRISPR/Cas9 systems have been successfully used for RAB11FIP4 knockout and provide complete protein elimination, ideal for antibody validation

  • siRNA approaches offer temporary knockdown and should employ at least two different siRNA sequences targeting different regions

  • For either approach, verify target reduction at both mRNA (RT-qPCR) and protein (Western blot with validated antibodies) levels

• Guide RNA/siRNA design considerations:

  • Ensure target sequences are specific to RAB11FIP4B and do not affect other RAB11FIP family members

  • Verify that target sequences do not contain SNPs that might affect efficiency

  • When validating antibodies that may recognize multiple isoforms, design knockdown strategies that target all relevant isoforms

• Control selection and experimental design:

  • Include proper negative controls (non-targeting gRNA/siRNA)

  • Use positive controls (knockdown of housekeeping genes with validated phenotypes)

  • For antibody validation, a reduction of at least 25% in target protein level is considered significant

• Cell line considerations:

  • Select cell lines with detectable endogenous expression of RAB11FIP4B

  • Consider using cell line panels with varying expression levels to test antibody sensitivity

  • Include cell types relevant to the intended experimental application (e.g., proximal tubule cells for cystinosis studies)

• Validation assessment criteria:

  • For Western blot: the band of interest should show significant reduction while non-specific bands remain unchanged

  • For immunofluorescence: specific signal should decrease while background remains constant

  • Quantify results using appropriate software to document the degree of signal reduction

Research has shown that genetic validation approaches are particularly important for immunofluorescence applications, where orthogonal validation methods may not accurately predict antibody performance . When validating RAB11FIP4B antibodies, researchers should be aware that knockout of this protein may affect cell growth, invasion, and metastasis in certain cell types, potentially complicating long-term experiments .

How can RAB11FIP4B antibodies be used to investigate its role in vesicular trafficking pathways?

Investigating RAB11FIP4B's role in vesicular trafficking requires strategic application of antibodies in combination with trafficking assays:

• Cargo trafficking assays:

  • Track internalization and recycling of classic cargo proteins (transferrin, integrins, receptor tyrosine kinases)

  • Use pulse-chase approaches with fluorescently labeled cargo

  • Combine with RAB11FIP4B antibody staining to correlate trafficking defects with RAB11FIP4B localization

  • Quantify colocalization of RAB11FIP4B with cargo at different time points during trafficking

• Endosomal compartment analysis:

  • Use RAB11FIP4B antibodies in combination with compartment markers:

    • Early endosomes (EEA1, Rab5)

    • Recycling endosomes (Rab11)

    • Late endosomes/lysosomes (LAMP1, LAMP2A)

  • Implement live-cell imaging with compartment markers to track RAB11FIP4B dynamics

  • Quantify changes in endosomal morphology, distribution, and number after RAB11FIP4B manipulation

• Specialized trafficking pathways:

  • Examine RAB11FIP4B's role in specialized contexts:

    • Cytokinesis (localization to midbody and cleavage furrow)

    • Autophagy (effects on autophagosome levels and LC3B-II expression)

    • Chaperone-mediated autophagy (influence on LAMP2A localization)

    • Receptor recycling (impact on megalin distribution in proximal tubule cells)

• Membrane recruitment dynamics:

  • Use antibodies to track RAB11FIP4B translocation to membranes under different conditions

  • Implement subcellular fractionation followed by Western blotting to quantify membrane association

  • Consider in vitro membrane binding assays with recombinant RAB11FIP4B and specific lipids

• Functional perturbation experiments:

  • Block RAB11FIP4B function using:

    • Function-blocking antibodies that target interaction domains

    • Expression of dominant-negative constructs

    • Rapid protein degradation systems (AID, PROTAC)

  • Monitor effects on trafficking pathways using live-cell imaging