rab11fip4a Antibody

What is RAB11FIP4A and what cellular functions does it regulate?

RAB11FIP4A (RAB11 Family Interacting Protein 4A) functions as a regulator of endocytic traffic by participating in membrane delivery. The protein is required for the abcission step in cytokinesis, possibly by acting as an 'address tag' delivering recycling endosome membranes to the cleavage furrow during late cytokinesis. Additionally, it may play a role in differentiation during retinal development .

RAB11FIP4 belongs to the family of Rab11 effector proteins associated with recycling endosomes. These proteins interact with RAB11, a small GTP-binding protein that regulates intracellular trafficking of recycling endosomes and is thereby involved in several neural functions . The interaction between RAB11 and its effector proteins like RAB11FIP4 is crucial for proper cellular trafficking and functions.

What experimental applications are RAB11FIP4A antibodies suitable for?

RAB11FIP4A antibodies are suitable for multiple experimental applications, with specificity varying by product. Based on available research tools, these antibodies can be used in:

For optimal results, antibody dilutions should be titrated for each experimental system and application. Product-specific recommendations should be consulted as performance can vary significantly between different antibodies targeting the same protein .

How should I select between polyclonal and monoclonal RAB11FIP4A antibodies?

The selection between polyclonal and monoclonal antibodies should be based on your specific experimental needs:

Polyclonal Antibodies:

• Recognize multiple epitopes on the target protein

• Generally provide higher sensitivity due to binding to multiple sites

• Better for detecting proteins with low expression levels

• More tolerant to minor protein denaturation or modification

• Show batch-to-batch variation that may affect reproducibility

• Example: The rabbit polyclonal antibody against zebrafish RAB11FIP4A is generated against a KLH-conjugated synthetic peptide from the central region (amino acids 183-216)

Monoclonal Antibodies:

• Recognize a single epitope

• Provide higher specificity and consistency

• Better for applications requiring batch-to-batch reproducibility

• May be more sensitive to protein modifications that alter the specific epitope

• Generally show less background and cross-reactivity

Recent research suggests that recombinant antibodies may outperform both traditional monoclonal and polyclonal antibodies. A study by Ayoubi et al. found that only around a third of polyclonal and monoclonal antibodies recognized their target in the experimental approaches they were recommended for, while recombinant antibodies showed better performance .

How can I validate the specificity of my RAB11FIP4A antibody?

Rigorous validation of antibody specificity is crucial for reliable experimental results. Recent studies highlight that many commercially available antibodies fail to recognize their intended targets . Follow these methodological approaches to validate RAB11FIP4A antibodies:

• Positive and Negative Controls:

  • Use cell lines with high RAB11FIP4A mRNA expression as positive controls

  • Employ CRISPR/Cas9 knockout systems to generate RAB11FIP4A-negative cells as definitive negative controls

  • Include tissues known to express or lack the target protein (based on search results, consider zebrafish tissue for RAB11FIP4A studies)

• Multiple Detection Methods:

  • Validate across at least three techniques (Western blot, immunofluorescence, and immunoprecipitation)

  • Manufacturers often validate only with Western blot, which is insufficient for comprehensive specificity assessment

• Peptide Competition Assay:

  • Pre-incubate the antibody with the immunizing peptide (if available)

  • This should abolish specific signals if the antibody is truly specific

• Orthogonal Methods:

  • Correlate antibody-based detection with mRNA expression data

  • Compare results with alternative antibodies targeting different epitopes of RAB11FIP4A

• Signal Localization Analysis:

  • Confirm that the detected signals match the expected subcellular localization

  • For RAB11FIP4A, appropriate localization would involve recycling endosomes and potentially enrichment at the cleavage furrow during cytokinesis

Third-party validation has proven valuable in identifying antibody specificity issues. Consider using antibodies that have undergone independent validation .

What are the critical differences between RAB11FIP4A, RAB11A, and other RAB11-associated proteins?

Understanding the relationships and distinctions between RAB11FIP4A and related proteins is essential for experimental design and interpretation:

In the adult mouse brain, RAB11A and RAB11B show similar expression in most neurons, but RAB11A also shows weak signals in the neuropil. In cultured neurons, they display only partial co-localization and differential distribution in both soma and neurites, with RAB11A appearing more abundant at presynapses than RAB11B .

These differences suggest distinct functional roles despite structural similarities, emphasizing the importance of specific antibodies for distinguishing between these related proteins.

How do I optimize immunofluorescence protocols for detecting RAB11FIP4A in neuronal cells?

Optimizing immunofluorescence protocols for RAB11FIP4A detection in neuronal cells requires special consideration of the protein's localization and expression patterns:

• Fixation Method Selection:

  • For membrane-associated proteins like RAB11FIP4A, 4% paraformaldehyde (PFA) for 15-20 minutes at room temperature is generally recommended

  • Avoid methanol fixation which can disrupt membrane structures

  • Consider light fixation (2% PFA) if antigen accessibility is an issue

• Permeabilization Optimization:

  • Test different permeabilization reagents: 0.1-0.3% Triton X-100, 0.1% saponin, or 0.1% digitonin

  • Saponin or digitonin may better preserve membrane structures compared to Triton X-100

  • Duration: 5-10 minutes for Triton X-100; 15-30 minutes for saponin/digitonin

• Antigen Retrieval Considerations:

  • Heat-mediated antigen retrieval in citrate buffer (pH 6.0) or TE buffer (pH 9.0) may improve signal

  • Determine necessity through parallel experiments with and without retrieval

• Antibody Dilution and Incubation:

  • Start with manufacturer's recommended dilution (typically 1:200-1:800 for IF/ICC)

  • Extended primary antibody incubation (overnight at 4°C) often improves specific signal

  • Include proper washing steps (3-5 times, 5 minutes each) with PBS containing 0.1% Tween-20

• Co-staining Strategies:

  • Co-stain with established markers of recycling endosomes (e.g., RAB11A/B)

  • Include markers for subcellular compartments (EEA1 for early endosomes; TGN46 for trans-Golgi)

  • For developmental studies, consider co-staining with neuronal maturation markers

• Signal Amplification:

  • For low abundance proteins, consider tyramide signal amplification

  • Biotin-streptavidin systems can enhance detection sensitivity

• Mounting and Imaging:

  • Use anti-fade mounting medium to prevent photobleaching

  • Consider super-resolution microscopy for detailed localization studies

For cultured neurons specifically, timing is crucial as RAB11FIP4A distribution may change during neuronal development. Consider analyzing expression at different developmental stages (DIV1, DIV7, DIV14, DIV21) to capture dynamic changes in localization patterns.

Why might I observe multiple bands when using RAB11FIP4A antibodies in Western blotting?

Multiple bands in Western blotting when using RAB11FIP4A antibodies can result from several factors:

• Post-translational Modifications:

  • Phosphorylation, glycosylation, or ubiquitination may cause mobility shifts

  • Different tissue/cell types may exhibit varying modification patterns

• Alternative Splicing:

  • RAB11FIP4 may have multiple isoforms due to alternative splicing

  • The antibody might recognize epitopes present in multiple splice variants

• Protein Degradation:

  • Partial degradation during sample preparation can produce fragments

  • Add protease inhibitors freshly to lysis buffers

  • Maintain samples at 4°C during preparation

• Cross-Reactivity:

  • The antibody may cross-react with related proteins (other RAB11FIPs)

  • Antibodies raised against human RAB11FIP4 might detect both RAB11FIP4A and other isoforms in certain species

• Non-specific Binding:

  • Insufficient blocking or too high antibody concentration

  • Try 5% non-fat dry milk or 3-5% BSA in TBS-T for blocking

  • Increase washing time/frequency with TBS-T

Troubleshooting Approaches:

• Perform peptide competition assays to identify specific bands

• Validate with siRNA/shRNA knockdown or CRISPR knockout samples

• Compare results with different antibodies targeting distinct epitopes

• Use gradient gels for better resolution of closely migrating bands

• Optimize sample denaturing conditions (temperature, reducing agent concentration)

The expected molecular weight of RAB11FIP4A may vary by species, so consult literature specific to your experimental model. For human RAB11FIP4, the calculated molecular weight is approximately 71 kDa .

What controls should I include when using RAB11FIP4A antibodies for co-immunoprecipitation studies?

Co-immunoprecipitation (Co-IP) experiments with RAB11FIP4A antibodies require rigorous controls to ensure reliable results:

• Essential Controls:

  a. Input Control:

  • Run 5-10% of the lysate used for IP to confirm protein expression in starting material

  b. Negative Control Antibody:

  • IgG from the same species and at the same concentration as the RAB11FIP4A antibody

  • Should not pull down RAB11FIP4A or interacting proteins

  c. Reciprocal IP:

  • If studying interaction with RAB11A/B, perform reverse IP using RAB11A/B antibodies

  • Both approaches should confirm the interaction

  d. Knockout/Knockdown Control:

  • Use lysates from cells with RAB11FIP4A depletion to confirm specificity

• Advanced Controls:

  a. GTPase State Controls:

  • Since RAB proteins interact with effectors in a GTP-dependent manner

  • Include GDP-locked (inactive) and GTP-locked (active) RAB11 mutants

  b. Interaction Domain Mutants:

  • Test RAB11FIP4A constructs with mutations in the RAB11-binding domain

  c. Crosslinking Controls:

  • If using crosslinking, include non-crosslinked samples

  • Titrate crosslinker concentration to optimize specific interactions

• Technical Considerations:

  Parameter	Recommendation	Rationale
  Lysis Buffer	RIPA or NP-40 with protease inhibitors	Balance between protein solubilization and preserving interactions
  Antibody Amount	0.5-4.0 μg per 1-3 mg lysate	Sufficient for efficient pulldown without non-specific binding
  Incubation Time	2 hours to overnight at 4°C	Allows sufficient binding time while minimizing degradation
  Washing Stringency	3-5 washes with increasing salt concentration	Reduces background while preserving specific interactions

• Validation Approaches:

  • Confirm interactions using alternative techniques (proximity ligation assay, FRET)

  • Compare results under different cellular conditions (serum starvation, growth factor stimulation)

  • Test interaction in different cell types/tissues as expression levels of adaptor proteins may vary

When reporting results, include blots of all controls and clearly state washing conditions and buffer compositions to ensure reproducibility.

How can I distinguish between RAB11FIP4A and other RAB11FIP family members in my experiments?

Distinguishing between RAB11FIP4A and other RAB11FIP family members requires careful experimental design:

• Antibody Selection Strategies:

  • Choose antibodies raised against non-conserved regions of RAB11FIP4A

  • Target the C-terminal region which typically shows greater sequence divergence

  • Validate specificity using overexpression systems for each RAB11FIP family member

• Western Blot Differentiation:

  • RAB11FIP proteins have different molecular weights:

    • RAB11FIP1: ~120 kDa

    • RAB11FIP2: ~55-60 kDa

    • RAB11FIP3: ~90-95 kDa

    • RAB11FIP4: ~71 kDa

    • RAB11FIP5: ~70 kDa

  • Use high-resolution SDS-PAGE (8-10% gels) with longer running times

• RT-qPCR for Gene Expression:

  • Design primers specific to non-conserved regions of RAB11FIP4A

  • Include melt curve analysis to confirm single amplification product

  • Compare expression patterns across tissues/cell types (different RAB11FIPs show distinct tissue distribution)

• Immunofluorescence Localization Patterns:

  • RAB11FIPs show subtly different subcellular localization:

    • Class I FIPs (RAB11FIP1/2/5): Contain C2 domains

    • Class II FIPs (RAB11FIP3/4): Contain EF-hand domains

  • Co-staining with organelle markers can help distinguish localization patterns

• Functional Assays:

  • RAB11FIP4A is specifically involved in cytokinesis and retinal development

  • Use functional readouts specific to RAB11FIP4A (e.g., cytokinesis defects)

  • Complement with siRNA-mediated knockdown of specific RAB11FIPs

• Mass Spectrometry Confirmation:

  • For definitive identification, immunoprecipitate the protein and perform mass spectrometry

  • Look for peptides unique to RAB11FIP4A not present in other family members

The RAB11FIP family is divided into Class I (RAB11FIP1, RAB11FIP2, RAB11FIP5) and Class II (RAB11FIP3, RAB11FIP4) based on their domain structure. Class I FIPs contain a C2 domain while Class II FIPs contain EF-hand domains. Understanding these structural differences can help inform experimental design to distinguish between family members.

How can RAB11FIP4A antibodies be used to study neurodevelopmental processes?

RAB11FIP4A antibodies offer valuable tools for investigating neurodevelopmental processes, particularly given the protein's potential role in retinal development :

• Developmental Expression Profiling:

  • Track RAB11FIP4A expression across developmental timepoints using immunoblotting

  • Perform immunohistochemistry on brain sections from different developmental stages

  • Compare expression patterns with established neurodevelopmental markers

• Neuronal Differentiation Studies:

  • Analyze RAB11FIP4A localization during differentiation of neural progenitors

  • Study co-localization with RAB11A/B, which show developmental stage-specific distribution patterns in the brain

  • Examine potential roles in growth cone dynamics and neurite extension

• Cell Division in Neural Progenitors:

  • Given RAB11FIP4A's role in cytokinesis , investigate its function in dividing neural stem cells

  • Analyze symmetric vs. asymmetric divisions using co-staining with polarity markers

  • Correlate RAB11FIP4A localization with cleavage furrow formation

• Retinal Development Models:

  • Study RAB11FIP4A in zebrafish models, where antibodies against the protein are available

  • Investigate potential roles in retinal cell differentiation and organization

  • Perform time-course immunofluorescence during retinal layer formation

• Methodology for Developmental Studies:

  • For tissue sections: Use fresh-frozen rather than paraffin-embedded tissue when possible

  • For embryonic tissue: Optimize fixation time (typically shorter than for adult tissue)

  • Include co-staining with markers for:

    • Neural progenitors (Sox2, Nestin)

    • Immature neurons (DCX, β-III-tubulin)

    • Mature neurons (NeuN, MAP2)

• Functional Approaches:

  • Combine antibody detection with manipulations of RAB11FIP4A expression

  • Design rescue experiments in knockdown/knockout models

  • Compare phenotypes with RAB11A/B perturbations, as these proteins show distinct distribution patterns in developing cortex

Recent research on RAB11A and RAB11B distribution indicates that RAB11A is enriched in the apical endfeet of apical radial glial cells in the developing cortex, while RAB11B is abundantly expressed in postmigratory neurons of the cortical plate . RAB11FIP4A's interaction with these proteins during development may provide insights into neuronal migration, polarization, and synaptogenesis.

What insights can RAB11FIP4A antibody studies provide about recycling endosome function in specialized cell types?

RAB11FIP4A antibody studies can reveal critical insights about recycling endosome function in specialized cell types:

• Neuronal Recycling Endosome Dynamics:

  • RAB11A appears more abundant at presynapses than RAB11B , suggesting specialized roles

  • RAB11FIP4A antibodies can help determine if this effector shows similar specialized distribution

  • Study potential roles in:

    • Synaptic vesicle recycling

    • Neurotransmitter receptor trafficking

    • Activity-dependent synapse modification

• Epithelial Cell Polarity:

  • Investigate RAB11FIP4A's role in apical-basolateral sorting

  • Analyze co-localization with polarity markers (E-cadherin, ZO-1)

  • Compare with RAB11A distribution, which shows enrichment in specific cellular domains

• Immune Cell Function:

  • Given findings about RAB11FIP5's role in NK cells , investigate RAB11FIP4A in immune cells

  • Study potential involvement in:

    • Cytokine secretion

    • Immunological synapse formation

    • Receptor recycling during activation

• Methodology for Cell-Type Specific Analysis:

  Cell Type	Recommended Approach	Key Markers for Co-localization
  Neurons	Primary cultures or brain sections	Synaptic markers (synaptophysin, PSD-95)
  Epithelial Cells	Polarized monolayers (MDCK cells)	Apical/basolateral markers (Na+/K+-ATPase, E-cadherin)
  Immune Cells	Isolated primary cells or cell lines	Activation markers (CD69, CD25)
  Dividing Cells	Synchronized cultures	Mitotic markers (phospho-histone H3)

• Advanced Imaging Approaches:

  • Live-cell imaging of fluorescently tagged RAB11FIP4A

  • Super-resolution microscopy to resolve endosomal subdomains

  • FRAP (Fluorescence Recovery After Photobleaching) to study dynamics

  • Correlative light and electron microscopy for ultrastructural localization

• Pathological Contexts:

  • Investigate RAB11FIP4A distribution in neurodevelopmental disorders

  • Compare with RAB11A/B, which are associated with similar neurodevelopmental disorders

  • Study potential alterations in cancer cells, particularly those with cytokinesis defects

Understanding the differential distribution and function of RAB11FIP4A compared to other RAB11 effectors can provide insights into the specialized roles of recycling endosomes in different cellular contexts.

How can I design experiments to study the interaction between RAB11FIP4A and RAB11A/B using antibodies?

Designing experiments to study RAB11FIP4A-RAB11A/B interactions requires careful consideration of both proteins' properties:

• Co-immunoprecipitation (Co-IP) Strategy:

  • Perform reciprocal Co-IPs using both RAB11FIP4A and RAB11A/B antibodies

  • Include GTPγS (activating) or GDP (inactivating) in lysates to test nucleotide-dependence

  • Use cell-permeable crosslinkers (DSP) to stabilize transient interactions

  • Recommended protocol:

    1. Lyse cells in buffer containing 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1% NP-40, protease inhibitors

    2. Pre-clear lysates with protein A/G beads

    3. Incubate with 2-4 μg antibody overnight at 4°C

    4. Add protein A/G beads for 2 hours

    5. Wash 4-5 times with decreasing detergent concentrations

    6. Elute and analyze by immunoblotting

• Proximity Ligation Assay (PLA):

  • Provides in situ detection of protein interactions

  • Use primary antibodies from different species (e.g., rabbit anti-RAB11FIP4A and mouse anti-RAB11A)

  • Signal indicates proteins are within 40 nm of each other

  • Compare signal distribution with conventional co-localization analysis

• FRET/FLIM Analysis:

  • Label RAB11FIP4A and RAB11A/B with appropriate fluorophore pairs

  • Measure energy transfer as indication of direct interaction

  • Analyze in fixed cells using antibodies conjugated to FRET-compatible fluorophores

• Domain Mapping:

  • Use antibodies targeting different regions of RAB11FIP4A

  • Compare interaction efficiency with RAB11A vs RAB11B

  • Design truncation constructs to identify minimal interaction domains

• Spatiotemporal Dynamics:

  • Study interaction during specific cellular processes:

    • Cell division (focus on midbody during cytokinesis)

    • Neuronal differentiation

    • Membrane recycling events

• Comparative Analysis with Other RAB11FIPs:

  • Include RAB11FIP1 as comparison (antibodies available )

  • Determine if RAB11FIP4A shows preferences for RAB11A vs RAB11B, given their distinct distribution patterns

• Functional Readouts:

  • Transferrin recycling assays

  • E-cadherin recycling in epithelial cells

  • AMPA receptor recycling in neurons

  • Cytokinesis completion time

Recent research shows that RAB11A and RAB11B have distinct distribution patterns in the brain and other organs . This suggests they may interact differently with effector proteins like RAB11FIP4A. By carefully designing interaction studies, you can determine if RAB11FIP4A shows preferential binding to either RAB11 isoform in different cellular contexts, potentially explaining their distinct functions.

How might RAB11FIP4A antibodies contribute to understanding neurodevelopmental disorders?

RAB11FIP4A antibodies could significantly advance our understanding of neurodevelopmental disorders through several research avenues:

• Expression Analysis in Patient Samples:

  • Compare RAB11FIP4A expression and localization in post-mortem brain tissues

  • Examine iPSC-derived neurons from patients with neurodevelopmental disorders

  • Study expression in organoids modeling neurodevelopmental conditions

• Connection to RAB11A/B Pathology:

  • Recent research indicates that pathogenic variants in RAB11A and RAB11B are associated with similar neurodevelopmental disorders

  • RAB11FIP4A antibodies can help determine if this effector's dysfunction contributes to similar phenotypes

  • Investigate if RAB11FIP4A distribution is altered in models with RAB11A/B mutations

• Developmental Trajectory Analysis:

  • Track RAB11FIP4A expression across neurodevelopmental timepoints in models of:

    • Autism spectrum disorders

    • Intellectual disability

    • Epilepsy

  • Compare with normal developmental trajectories

• Functional Studies in Disease Models:

  • Investigate RAB11FIP4A's role in neuronal migration defects

  • Study potential contributions to synaptic abnormalities

  • Examine links to cytoskeletal organization during neurite growth

• Therapeutic Target Potential:

  • Use antibodies to identify critical domains for RAB11FIP4A function

  • Develop screening assays for compounds that modulate RAB11FIP4A-RAB11 interactions

  • Explore the potential for normalizing recycling endosome function as a therapeutic strategy

• Methodological Considerations:

  • Use brain region-specific analysis to identify selective vulnerability

  • Combine with genetic models (knockin of patient mutations)

  • Employ quantitative image analysis for subtle distribution changes

Research has shown that RAB11A is enriched in apical radial glial cells and RAB11B in postmigratory neurons of the developing cortex . Disruption of these patterns could contribute to neurodevelopmental pathologies. RAB11FIP4A antibodies can help determine if this effector shows similar cell type-specific distribution and whether its mislocalization contributes to disease phenotypes.

What are the emerging techniques that could enhance RAB11FIP4A antibody applications in research?

Several emerging techniques could significantly enhance RAB11FIP4A antibody applications in research:

• Super-Resolution Microscopy:

  • Techniques such as STORM, PALM, and STED can resolve RAB11FIP4A localization at nanometer scale

  • Enables visualization of endosomal subdomains not visible with conventional microscopy

  • Can determine precise spatial relationships with RAB11A/B, which show only partial co-localization in neurons

• Expansion Microscopy:

  • Physical expansion of specimens allows super-resolution imaging on standard microscopes

  • Can reveal fine details of RAB11FIP4A distribution in complex cellular structures

  • Particularly valuable for analyzing dense neuronal structures

• Live-Cell Single-Molecule Imaging:

  • Track individual RAB11FIP4A molecules using antibody fragments or nanobodies

  • Study dynamics during trafficking events and cytokinesis

  • Correlate with RAB11A/B movements, which show differential distribution in neurons

• Antibody-Based Proximity Labeling:

  • Antibodies conjugated to enzymes like APEX2 or TurboID

  • Enables identification of proteins in close proximity to RAB11FIP4A

  • Can reveal cell type-specific interactomes

• Spatially-Resolved Transcriptomics:

  • Combine RAB11FIP4A antibody staining with in situ sequencing

  • Correlate protein localization with local mRNA expression

  • Identify potential regulatory relationships

• Cryo-Electron Tomography:

  • Immunogold labeling of RAB11FIP4A for ultrastructural localization

  • 3D visualization of endosomal compartments at molecular resolution

  • Determine precise membrane association patterns

• Optogenetic Manipulation Combined with Imaging:

  • Light-inducible dimerization systems to recruit RAB11FIP4A to specific locations

  • Monitor subsequent effects on trafficking using antibody detection

  • Test functional relationships with RAB11A vs RAB11B

• Microfluidic Devices:

  • Study RAB11FIP4A dynamics in controlled microenvironments

  • Analyze responses to specific stimuli in real-time

  • Particularly useful for neuronal compartmentalization studies

These advanced techniques can provide unprecedented insights into RAB11FIP4A biology, particularly when used in combination. For example, combining super-resolution microscopy with proximity labeling could reveal both the nanoscale organization of RAB11FIP4A and its molecular neighbors, helping to unravel the complex functions of this protein in various cellular contexts.