FIPS5 Antibody

Basic Research Considerations

• What is RAB11FIP5 and what cellular functions does it regulate?

  RAB11FIP5 (also known as Rip11, GAF1) is an effector protein that interacts with the small GTPase Rab11 and is critically involved in protein trafficking from apical recycling endosomes to the plasma membrane. It functions in:

  • Regulating endosomal recycling pathways

  • Facilitating insulin granule exocytosis

  • Modulating V-ATPase intracellular transport in response to extracellular acidosis

  • Ensuring important signaling molecules reach the cell surface efficiently

  In immune contexts, RAB11FIP5 shows elevated expression in individuals who develop broadly neutralizing antibodies (bnAbs) against HIV-1, with the highest differential expression observed in natural killer (NK) cells. This expression correlates with NK cell dysfunction and altered functionality, suggesting RAB11FIP5 plays a regulatory role in humoral immune responses .

• What is the molecular weight of RAB11FIP5 protein and how does this impact antibody detection?

  RAB11FIP5 has a calculated molecular weight of approximately 70 kDa, though observed molecular weights may vary:

  Source	Calculated MW	Observed MW	Possible Explanation
  Proteintech data	70 kDa	70 kDa	Native protein
  Abcam data	70 kDa	77 kDa	Post-translational modifications
  Boster Bio data	70.4 kDa	Varies by sample	Tissue-specific modifications

  When performing Western blot analysis, researchers should be aware that post-translational modifications, splice variants, or degradation products may affect the observed molecular weight. For optimal detection, use positive controls (such as transfected HEK-293T cells) alongside experimental samples and verify specificity using knockdown/knockout controls .

• Which tissue types and cell lines show strong RAB11FIP5 expression for positive controls?

  For effective experimental controls, the following tissues and cell lines have demonstrated reliable RAB11FIP5 expression:

  Tissues with confirmed expression:

  • Mouse kidney tissue

  • Mouse lung tissue

  • Mouse heart tissue

  • Rat heart tissue

  • Human cervical tissue

  Cell lines with confirmed expression:

  • A431 cells

  • HeLa cells

  • HEK-293T cells (especially useful for overexpression studies)

  When establishing baseline expression, consider using multiple tissue/cell types as RAB11FIP5 expression varies significantly across tissues. Natural killer (NK) cells show particularly high differential expression of RAB11FIP5 in HIV-1 infected individuals who develop broadly neutralizing antibodies compared to those who don't .

Methodology and Protocols

• What are the optimal sample preparation methods for Western blot detection of RAB11FIP5?

  Successful Western blot detection of RAB11FIP5 requires careful sample preparation:

  1. Lysis buffer selection: Use RIPA buffer supplemented with protease inhibitors for most applications. For membrane-associated protein interactions, consider NP-40 or Triton X-100 based buffers to preserve protein complexes.

  2. Denaturation conditions: Heat samples at 95°C for 5 minutes in Laemmli buffer containing 5% β-mercaptoethanol.

  3. Gel percentage: Use 8-10% SDS-PAGE gels for optimal separation near the 70 kDa range.

  4. Transfer conditions: Semi-dry transfer at 15V for 60 minutes or wet transfer at 100V for 60-90 minutes with methanol-containing buffer.

  5. Antibody dilutions: Primary antibody dilutions range from 1:1000-1:6000 depending on the source (see table) :

  Antibody Source	Recommended WB Dilution	Incubation Conditions
  Proteintech (14594-1-AP)	1:1000-1:6000	Overnight at 4°C
  Abcam (ab68947)	1:500	1-2 hours at RT or overnight at 4°C
  Boster Bio (A07274)	1:500-1:2000	Overnight at 4°C

• What controls should be implemented to validate RAB11FIP5 antibody specificity?

  Proper validation of RAB11FIP5 antibody specificity is crucial for generating reliable data:

  1. Positive controls:

     • Transfected cell lines overexpressing RAB11FIP5 (confirmed by Abcam with 293T cells)

     • Tissues with known high expression (mouse kidney, lung, A431 cells)

  2. Negative controls:

     • Knockout/knockdown validation using CRISPR/Cas9 or siRNA approaches

     • Secondary antibody-only controls to assess background

     • Blocking peptide competition assays (where available)

  3. Cross-validation:

     • Use multiple antibodies targeting different epitopes of RAB11FIP5

     • Confirm results using complementary techniques (IF/IHC for localization, WB for expression)

  A study investigating RAB11FIP5 in telencephalon development used CRISPR/Cas9 to generate knockout embryos, confirming specificity through DNA sequencing of the targeted genomic region that showed nucleotide deletions near the PAM region . This approach provides the strongest validation of antibody specificity.

• What are the optimal fixation and antigen retrieval methods for immunohistochemistry with RAB11FIP5 antibodies?

  Successful immunohistochemical detection of RAB11FIP5 requires appropriate fixation and antigen retrieval:

  Fixation protocols:

  • 4% paraformaldehyde (PFA) for 24-48 hours for tissue sections

  • 4% PFA for 15-20 minutes for cultured cells

  • Alcohol-based fixatives may preserve antigenicity better for some applications

  Antigen retrieval methods:
  According to Proteintech data, optimal results are achieved with:

  • Primary recommendation: TE buffer pH 9.0 with heat-induced epitope retrieval

  • Alternative method: Citrate buffer pH 6.0

  Blocking conditions:

  • 5-10% normal serum (species of secondary antibody) with 0.1-0.3% Triton X-100

  • 1-2 hour incubation at room temperature

  Antibody dilutions for IHC:

  • 1:250-1:1000 dilution range (Proteintech 14594-1-AP)

  Tissue-specific optimization may be necessary, as RAB11FIP5 detection varies across different samples. For dual-labeling studies, careful selection of compatible antibodies and detection systems is essential to avoid cross-reactivity.

Advanced Research Applications

• How does RAB11FIP5 expression correlate with NK cell dysfunction in HIV-1 infection?

  RAB11FIP5 expression shows a significant correlation with NK cell dysfunction in HIV-1 infection, particularly in individuals who develop broadly neutralizing antibodies (bnAbs):

  1. Expression pattern differences:

     • NK cells from bnAb-producing individuals exhibit the highest differential expression of RAB11FIP5 compared to individuals who don't produce bnAbs

     • RAB11FIP5 transcripts positively correlate with HIV-1 antibody neutralization breadth (r = 0.50, p ≤ 0.05; Spearman)

  2. NK cell subset alterations:

     • bnAb individuals show decreased CD56dim NK cell subsets

     • Increased CD56-CD16+ NK cell populations correlate with RAB11FIP5 expression

     • Negative correlation observed between CD56dim subset frequency and RAB11FIP5 mRNA levels

  3. Functional implications:

     • NK cells from bnAb individuals exhibit a more adaptive/dysfunctional phenotype

     • Impaired degranulation and cytokine production correlate with RAB11FIP5 transcript levels

     • Lower expression of killing molecules (GZMA, GZMK) and TRAIL in NK cells from bnAb individuals

  4. Methodological approach for investigating this correlation:

     • Isolate NK cells using negative selection or FACS sorting (CD3-CD56+CD16+)

     • Measure RAB11FIP5 expression by qPCR or Western blot

     • Assess NK cell functionality through degranulation assays (CD107a) and cytokine production

     • Analyze NK cell subset distribution using flow cytometry with CD56 and CD16 markers

  This data suggests that RAB11FIP5 may be a critical regulator of NK cell-mediated control of antibody responses, particularly in the context of chronic viral infections.

• What mechanisms link RAB11FIP5 to the generation of broadly neutralizing antibodies against HIV-1?

  Several interconnected mechanisms link RAB11FIP5 to broadly neutralizing antibody (bnAb) generation in HIV-1 infection:

  1. NK cell dysfunction pathway:

     • Elevated RAB11FIP5 expression correlates with emergence of dysfunctional NK cell subsets (CD56-CD16+)

     • NK cells from bnAb individuals show decreased expression of PLZF, FcεRγ, and Siglec7

     • These phenotypic changes are associated with impaired NK cell regulatory function

  2. Immunoregulatory connection:

     • NK cells normally constrain antibody responses through:

       • Lysis of CD4 T cells

       • Reduction of T follicular helper (Tfh) cell availability

       • Modulation of germinal center reactions

     • RAB11FIP5-associated NK dysfunction may release these constraints, permitting development of more diverse antibody responses

  3. Endosomal recycling effects:

     • RAB11FIP5 regulates recycling endosomal transport

     • This transport system controls surface expression of receptors on immune cells

     • Altered recycling may affect antigen presentation, B cell receptor signaling, or T cell help

  4. Experimental approach to investigate this mechanism:

     • Overexpress RAB11FIP5 in NK cells to confirm causality in NK dysfunction

     • Perform co-culture experiments with B cells and T follicular helper cells

     • Assess germinal center reactions in animal models with NK-specific RAB11FIP5 manipulation

     • Analyze B cell receptor trafficking in the context of RAB11FIP5 expression

  The study by Bradley et al. demonstrates that RAB11FIP5 is not merely a correlate but plays a functional role, as RAB11FIP5 overexpression directly modulated NK cell function, suggesting a causal relationship in the development of bnAbs during HIV-1 infection .

• How can RAB11FIP5 antibodies be used to study developmental processes involving ephrinB signaling?

  RAB11FIP5 antibodies can be strategically employed to investigate ephrinB signaling in developmental processes, particularly in telencephalon development:

  1. Co-immunoprecipitation (Co-IP) studies:

     • RAB11FIP5 interacts with ephrinB ligands through their highly conserved intracellular domains

     • Co-IP analysis using ephrinB-HA tagged proteins confirmed interactions between RAB11FIP5 and all ephrinB ligands

     • Protocol approach: Use anti-HA antibodies to pull down ephrinB complexes, then probe with RAB11FIP5 antibodies

  2. Spatiotemporal expression analysis:

     • Immunohistochemistry with RAB11FIP5 antibodies reveals expression patterns during development

     • Compare RAB11FIP5 and ephrinB localization in developing tissues

     • Assess co-localization in recycling endosomes using confocal microscopy

  3. Functional studies:

     • Use RAB11FIP5 antibodies to track protein dynamics after ephrinB activation

     • Investigate endocytosis and recycling of ephrinB receptors using pulse-chase experiments

     • Monitor changes in RAB11FIP5-ephrinB interactions during key developmental events

  4. Loss-of-function experiments:

     • CRISPR/Cas9 knockout of RAB11FIP5 (targeting first exon) to study effects on ephrinB signaling

     • Validate knockouts through DNA sequencing showing nucleotide deletions near PAM region

     • Analyze developmental phenotypes using transgenic embryos (e.g., tubb2b:mapt-GFP for neural tissues)

  The interplay between RAB11FIP5 and ephrinB signaling represents an important regulatory mechanism in embryonic development, particularly in neural tissues. RAB11FIP5 antibodies provide tools to dissect the trafficking of ephrinB ligands through recycling endosomes, potentially influencing gradient formation and signaling range during development.

• What are the technical challenges in distinguishing RAB11FIP5 from other RAB11 family-interacting proteins in research applications?

  Distinguishing RAB11FIP5 from other RAB11 family-interacting proteins (RAB11FIPs) presents several technical challenges requiring careful methodological approaches:

  1. Structural and sequence homology issues:

     • Five RAB11FIPs (FIP1, FIP2, FIP3, FIP4, FIP5) share conserved C-terminal Rab11-binding domains

     • Class I FIPs (FIP1, FIP2, FIP5) contain similar C2 domains

     • Antibody cross-reactivity can occur due to shared epitopes

  2. Validation strategies:

     • Western blot analysis using knockout/knockdown controls for each FIP family member

     • Comparison of migration patterns (FIP5: ~70 kDa; other FIPs have distinct molecular weights)

     • Use of multiple antibodies targeting different epitopes unique to RAB11FIP5

  3. Experimental approaches for specific detection:

     Technique	Approach	Challenge	Solution
     Immunoblotting	Use highly specific antibodies	Cross-reactivity	Verify with recombinant protein controls
     qPCR	Design primers to unique regions	Splice variants	Target exon boundaries specific to RAB11FIP5
     Immunostaining	Double-labeling with markers	Overlapping localization	Super-resolution microscopy
     Immunoprecipitation	Pull-down experiments	Co-precipitation of complexes	Mass spectrometry validation

  4. Subcellular localization differentiation:

     • RAB11FIP5 shows distinct localization to apical recycling endosomes

     • Use co-localization studies with compartment-specific markers

     • Perform fractionation experiments to isolate specific endosomal populations

  Researchers should carefully validate antibody specificity using knockout controls and recombinant protein standards. When possible, complementary approaches (genetic manipulation, transcript analysis) should be used alongside antibody-based detection to confirm specificity for RAB11FIP5 versus other RAB11FIP family members.

Methodological Considerations for Specialized Applications

• How should researchers design immunoprecipitation experiments to study RAB11FIP5 protein-protein interactions?

  Optimized immunoprecipitation (IP) protocols for RAB11FIP5 interaction studies require careful consideration of several factors:

  1. Lysis buffer optimization:

     • Use mild non-ionic detergents (0.5-1% NP-40 or 1% Triton X-100)

     • Include protease inhibitors, phosphatase inhibitors, and 1-2 mM EDTA

     • Buffer composition: 50 mM Tris-HCl pH 7.4, 150 mM NaCl, 1 mM EDTA

     • For membrane protein interactions, consider using digitonin-based buffers

  2. IP strategy selection:

     • Direct IP: Use anti-RAB11FIP5 antibodies coupled to protein A/G beads

     • Co-IP: Use antibodies against suspected interaction partners

     • Tagged protein approach: Express tagged RAB11FIP5 (HA, FLAG, or GFP tags)

  3. Case study - ephrinB interaction:
     Researchers successfully identified RAB11FIP5 as an ephrinB-interacting protein using:

     • ephrinB2-HA fusion proteins for IP

     • Mass spectrometry analysis of immune complexes

     • Validation using reciprocal co-IP with multiple ephrinB ligands

     • This approach revealed that RAB11FIP5 interacts with all ephrinB ligands through their conserved intracellular domains

  4. Washing and elution conditions:

     • Use increasing stringency washes to reduce non-specific binding

     • Consider crosslinking antibodies to beads to prevent antibody contamination

     • Elute with gentle conditions to preserve protein complexes (glycine pH 2.8 or competitive elution)

  5. Confirmation strategies:

     • Reverse co-IP experiments (using antibodies against different complex components)

     • Mass spectrometry analysis of immunoprecipitated complexes

     • Proximity labeling approaches (BioID or APEX) as complementary methods

  When analyzing RAB11FIP5 interactions with membrane trafficking machinery, researchers should be particularly attentive to membrane solubilization conditions to preserve physiologically relevant protein complexes.

• What are the key considerations for studying RAB11FIP5 in primary immune cells?

  Investigating RAB11FIP5 in primary immune cells presents unique challenges requiring specialized approaches:

  1. Cell isolation and handling:

     • For NK cells (which show highest differential RAB11FIP5 expression in HIV studies):

       • Use negative selection to avoid receptor triggering

       • Isolate CD3-CD56+CD16+ NK cells by FACS or magnetic separation

       • Process samples quickly to prevent ex vivo changes in RAB11FIP5 expression

     • For broader immune cell profiling:

       • Isolate PBMCs using density gradient centrifugation

       • Further separate into CD19+ B cells, CD4+ T cells, CD8+ T cells, and non-B/T cells

       • Further fractionate non-B/T cells into CD14+ monocytes, CD56+CD16+ NK cells, and dendritic cell populations

  2. Expression analysis approaches:

     • qPCR shows highest differential expression of RAB11FIP5 in NK cells from HIV-1 bnAb individuals

     • RNA-seq of purified NK cells identified RAB11FIP5 among 95 differentially expressed transcripts

     • Flow cytometry can assess RAB11FIP5 protein levels in conjunction with cell surface markers

  3. Functional assessments:

     • NK cell functional assays:

       • Degranulation (CD107a expression)

       • Cytokine production (IFN-γ, TNF-α)

       • Cytotoxicity against target cells

     • Correlation of function with RAB11FIP5 expression levels

     • Assessment of recycling endosome function using transferrin recycling assays

  4. Sample requirements:
     When designing experiments with primary human cells, consider:

     • Typically require 10-20 mL of peripheral blood for sufficient NK cell isolation

     • Fresh samples yield better results than cryopreserved cells for functional studies

     • Matched controls are essential due to high donor-to-donor variability

  The Bradley et al. study demonstrated that NK cells from bnAb individuals had altered functionality that correlated with RAB11FIP5 transcript levels, suggesting that RAB11FIP5 overexpression directly modulates NK cell function in ways that permit bnAb development .