rab11fip3 Antibody

Basic Research Considerations

• What is RAB11FIP3 and what are the optimal applications for RAB11FIP3 antibodies?

RAB11FIP3 (RAB11 family interacting protein 3) is a downstream effector molecule for Rab11 GTPase involved in endocytic trafficking, cytokinesis, and intracellular ciliogenesis. It functions as a dual effector for both Rab11 and Arf6 GTPases .

RAB11FIP3 antibodies are primarily used in the following applications:

For optimal results, each antibody should be titrated in your specific experimental system, as reactivity can vary based on sample preparation and experimental conditions .

• What storage conditions ensure maximum stability and performance of RAB11FIP3 antibodies?

RAB11FIP3 antibodies require specific storage conditions to maintain activity:

• Long-term storage: -20°C (stable for one year after shipment)

• Short-term storage: 4°C for up to one month

• Formulation: Most are supplied in PBS with 0.02% sodium azide and 50% glycerol, pH 7.3

• Aliquoting: Recommended for -20°C storage to avoid freeze-thaw cycles, though some formulations specifically note that aliquoting is unnecessary

When handling: Allow the antibody to equilibrate to room temperature before opening, and centrifuge if the solution is not completely clear . For 20μl sizes, note that they may contain 0.1% BSA in the formulation .

• How should researchers validate the specificity of RAB11FIP3 antibodies?

Validation of RAB11FIP3 antibody specificity requires multiple complementary approaches:

• Molecular weight verification: Although the predicted molecular weight of RAB11FIP3 is 82.4 kDa, it has been observed at ~120 kDa in human cell lysates and ~150 kDa in mouse cell lysates. An unexplained 40 kDa band has also been reported in HeLa and 293T cell lysates . These variations necessitate careful interpretation of Western blot results.

• Knockdown validation: Several publications have used siRNA knockdown to confirm antibody specificity. Two distinct siRNA oligos targeting RAB11FIP3 (such as FIP3#1 and FIP3#2 5'-aaggcgtgtgctggagctgga-3') should ideally be used to rule out off-target effects .

• Cross-reactivity testing: Test the antibody against multiple species and cell types to confirm the specificity claims. Some manufacturers verify specificity using protein arrays containing the target protein plus 383 other non-specific proteins .

• Multiple antibody comparison: When possible, compare results from different antibody clones targeting different epitopes of RAB11FIP3 .

• What antigen retrieval methods are recommended for RAB11FIP3 immunohistochemistry?

For optimal antigen retrieval in RAB11FIP3 immunohistochemistry:

For cell culture samples, fixation with 4% paraformaldehyde for 15 minutes followed by permeabilization with 0.4% saponin has shown successful results for immunofluorescence microscopy studies of RAB11FIP3 . For some specific applications, ice-cold methanol fixation has been reported to work well for FIP3 staining .

Advanced Research Applications

• How can researchers investigate RAB11FIP3's role in cancer cell motility and what phenotypes should be monitored?

To investigate RAB11FIP3's role in cancer cell motility, researchers should employ these methodological approaches:

Experimental design:

• RNA interference: Use siRNA-mediated knockdown of RAB11FIP3 in cancer cell lines (e.g., MDA-MB-231 breast carcinoma cells) . Ensure >90% transfection efficiency using optimized methods like Lipofectamine2000.

• Cell spreading assay: Place mock or FIP3 siRNA-treated cells onto ECM-coated (e.g., collagen) coverslips for defined periods (1-3 hours). Fix cells, stain with rhodamine-conjugated phalloidin, and quantify:

  • Surface area (outlined edges based on phalloidin staining)

  • Polarization ratio (length/width of the cell at its widest point)

• Monitor multiple phenotypes:

  • Cytoskeletal organization changes

  • Cell spreading kinetics

  • Directional persistence during migration

  • Focal adhesion dynamics

Research findings indicate that FIP3 is associated with breast cancer cell motility regulation through actin cytoskeleton modulation . Studies comparing RAB11FIP3 expression and function between cancerous (MDA-MB-231) and non-cancerous cell lines can provide valuable insights into its specific roles in malignant transformation .

• What is the significance of RAB11FIP3 in the Rab11-Rabin8-Rab8 ciliogenesis cascade and how can this be experimentally studied?

RAB11FIP3 plays a crucial role in the Rab11-Rabin8-Rab8 ciliogenesis cascade by:

• Facilitating the orderly assembly of a ciliary targeting complex containing:

  • Rab11

  • ASAP1

  • Rabin8/RAB3IP

  • RAB11FIP3

  • ARF4

• This complex directs preciliary vesicle trafficking to the mother centriole and initiates ciliogenesis .

• RAB11FIP3 specifically promotes the activity of both Rab11a and the Arf GTPase-activating protein ASAP1 in the ARF4-dependent Golgi-to-cilia transport of sensory receptors like rhodopsin .

Experimental approaches to study this process:

• Use siRNA-mediated knockdown of FIP3 to observe effects on ciliary formation and function

• Employ immunofluorescence to track localization changes of complex components during ciliogenesis

• Perform co-immunoprecipitation assays to confirm protein-protein interactions within the complex

• Implement live cell imaging to monitor the dynamics of vesicle trafficking during the process

Research has demonstrated that FIP3 coordinates the interactions of ASAP1 and Rab11a with Rabin8, shaping the binding pocket for Rabin8 within the ASAP1-Rab11a-FIP3 targeting complex . Without FIP3, ciliary targeting is abolished and leads to mislocalization of proteins like rhodopsin .

• How can researchers distinguish between actual and anomalous molecular weights of RAB11FIP3 in Western blot analysis?

Resolving molecular weight discrepancies for RAB11FIP3 requires systematic technical approaches:

The molecular weight puzzle:

• Calculated molecular weight: 82.4 kDa (756 amino acids)

• Observed in human lysates: ~120 kDa

• Observed in mouse lysates: ~150 kDa

• Unexplained 40 kDa band in HeLa and 293T lysates

Methodological approach to resolve discrepancies:

• Sample preparation optimization:

  • Use multiple lysis buffers with different detergent compositions

  • Include phosphatase inhibitors to preserve post-translational modifications

  • Test both reducing and non-reducing conditions

• Technical verification:

  • Run gradient gels (4-20%) to improve resolution

  • Include multiple molecular weight markers from different manufacturers

  • Perform peptide competition assays to confirm specificity of bands

• Biological validation:

  • Compare with recombinant RAB11FIP3 protein as a standard

  • Perform siRNA knockdown to identify which bands diminish

  • Compare expression patterns across multiple cell lines

These size variations likely reflect post-translational modifications or tissue-specific isoforms of RAB11FIP3. Researchers should always validate which band corresponds to their target by knockdown experiments before quantitative analysis .

• What experimental designs are optimal for investigating RAB11FIP3's role in cytokinesis?

To investigate RAB11FIP3's role in cytokinesis, implement this systematic experimental approach:

• Cellular depletion strategies:

  • siRNA-mediated knockdown (verify with Western blot)

  • CRISPR-Cas9 gene editing for complete knockout

  • Dominant-negative mutant expression

• Live cell imaging techniques:

  • Time-lapse microscopy with fluorescently-tagged RAB11FIP3

  • Monitor dynamics during different stages of cell division

  • Focus on telophase and abscission phases where RAB11FIP3 is most active

• Key phenotypes to quantify:

  • Timing of cytokinesis progression

  • Frequency of multinucleated cells

  • Midbody formation and resolution

  • Membrane trafficking to the cleavage furrow

Research findings indicate that RAB11FIP3 functions with Rab11 to transport vesicles derived from recycling endosomes to the cleavage furrow via centrosome-anchored microtubules during telophase . These vesicles deliver membrane components during late cytokinesis and abscission . The recruitment of RAB11FIP3-containing endosomes to the cleavage furrow and tethering to the midbody is co-mediated by RAB11FIP3 interaction with ARF6-exocyst and RACGAP1-MKLP1 tethering complexes .

• How can researchers effectively study the dynamic localization changes of RAB11FIP3 during cell cycle progression?

To track RAB11FIP3 localization throughout the cell cycle, implement this comprehensive approach:

Methodological framework:

• Cell synchronization techniques:

  • Double thymidine block for G1/S boundary

  • Nocodazole treatment for M-phase enrichment

  • Release from synchronization and collect time points across cell cycle

• Advanced imaging techniques:

  • Confocal microscopy with co-staining for cell cycle markers

  • Super-resolution microscopy for detailed subcellular localization

  • Live-cell imaging with fluorescently tagged RAB11FIP3

• Co-localization analysis:

  • Endosomal markers (early, recycling, sorting endosomes)

  • Centrosomal markers

  • Midbody components

  • Golgi apparatus markers

Cell cycle-specific localization patterns:

• Interphase: In vesicles continuously moving from peripheral sorting endosomes toward the pericentrosomal endosomal recycling compartment (ERC)

• Early mitosis: Diffuse distribution throughout the cell

• Anaphase onset: Sequestered to centrosomes at opposite poles of the cell

• Telophase: Vesicles move from centrosomes to the furrow, then to the midbody to aid in abscission

For optimal visualization in fixed cells, employ dual immunofluorescence strategies using antibodies against RAB11FIP3 with cell cycle phase-specific markers. For dynamic studies, stable cell lines expressing fluorescently-tagged RAB11FIP3 combined with other tagged markers enable real-time observation of localization changes during cell division .

• What approaches can be used to study RAB11FIP3's interaction with the dynein motor complex and its impact on cellular trafficking?

To investigate RAB11FIP3's interaction with the dynein motor complex:

Experimental design pipeline:

• Interaction validation:

  • Co-immunoprecipitation of RAB11FIP3 with dynein components

  • Proximity ligation assays to confirm interactions in situ

  • GST pull-down assays with purified components to identify direct binding regions

• Functional disruption approaches:

  • Express truncated RAB11FIP3 lacking dynein-binding domains

  • Use dynein inhibitors (e.g., ciliobrevin) to block motor function

  • siRNA knockdown of specific dynein components

• Dynamic trafficking analysis:

  • Live cell imaging with dual-labeled endosomes (RAB11FIP3 and cargo)

  • Single-particle tracking to measure directionality and velocity

  • Photoactivatable fluorescent proteins to track specific endosome populations

Key findings to investigate:
RAB11FIP3 is recruited by Rab11 to endosomes where it links Rab11 to the dynein motor complex . This functional Rab11-RAB11FIP3-dynein complex regulates the movement of peripheral sorting endosomes along microtubule tracks toward the microtubule organizing center/centrosome, generating the endocytic recycling compartment during interphase . Importantly, RAB11FIP3 facilitates the interaction between dynein and dynactin and activates dynein processivity—the ability to move along microtubules for long distances without detachment .

A comprehensive approach combining these methods will provide mechanistic insight into how RAB11FIP3 coordinates endosomal trafficking through dynein motor regulation.

Technical Troubleshooting

• What strategies can resolve non-specific binding issues when using RAB11FIP3 antibodies in immunofluorescence applications?

To minimize non-specific binding in RAB11FIP3 immunofluorescence:

Optimization protocol:

• Blocking optimization:

  • Test different blocking agents:

    • Standard: PBS with 0.2% BSA and 1% FBS

    • Alternative: 5% normal serum from the secondary antibody host species

    • Commercial blocking solutions with proprietary formulations

  • Extend blocking time to 1-2 hours at room temperature

• Antibody dilution optimization:

  • Titrate primary antibody (recommended range: 1-4 μg/ml)

  • For polyclonal antibodies, higher dilutions often reduce background

  • Consider using monoclonal antibodies for higher specificity

• Sample preparation refinement:

  • Fixation method comparison:

    • 4% paraformaldehyde (15 min) with 0.4% saponin permeabilization

    • Ice-cold methanol specifically reported to work well for FIP3 staining

  • Extensive washing between steps (minimum 3 × 5 minutes)

  • Use 0.1% Tween-20 in wash buffers to reduce non-specific interactions

• Controls to implement:

  • Include a no-primary antibody control

  • Use RAB11FIP3 siRNA knockdown samples as negative controls

  • Pre-absorb antibody with immunizing peptide if available

For challenging applications, antibody purification through antigen-affinity chromatography has proven successful with RAB11FIP3 antibodies , as has the validation of antibody specificity on protein arrays containing target protein plus numerous non-specific proteins .

• How can researchers differentiate between RAB11FIP3 and other RAB11 family interacting proteins in experimental systems?

To differentiate between RAB11FIP3 and other RAB11 family interacting proteins:

Multifaceted discrimination approach:

• Antibody selection:

  • Choose antibodies raised against unique epitopes of RAB11FIP3

  • Many successful antibodies target the C-terminal region

  • Verify antibody specificity against other FIP family members

• Molecular weight discrimination:

  • RAB11FIP3: 82.4 kDa (calculated), observed at ~120 kDa (human) or ~150 kDa (mouse)

  • Other FIPs have distinct molecular weights:

    • RAB11FIP1: ~71 kDa

    • RAB11FIP2: ~59 kDa

    • RAB11FIP5/Rip11: ~56 kDa

• Cellular localization patterns:

  • RAB11FIP3: Distinctly localizes to the midbody during cytokinesis

  • Use cellular contexts where different FIPs show unique distributions

• RNA interference with subtype specificity:

  • Design siRNAs targeting non-conserved regions

  • Compare phenotypes of RAB11FIP3 knockdown with other FIP family members

  • In breast cancer motility studies, RAB11FIP3 knockdown should be compared with Rip11/FIP5 knockdown to determine specific contributions

• RT-PCR for expression verification:

  • Use PCR primers specific to unique regions of RAB11FIP3

  • Compare expression profiles across different cell lines

  • For example, RT-PCR has been used to compare RAB11FIP3 expression in MDA-MB-231 and MCF-7 cells