rabl3 Antibody

What cellular functions does RABL3 regulate, and why is it important for research?

RABL3 serves multiple critical cellular functions that make it an important research target:

• Lymphopoiesis regulation: RABL3 is essential for proper lymphoid progenitor development. Mice with hypomorphic RABL3 mutations (such as the xiamen mutation) display profound defects in B cell, T cell, and natural killer (NK) cell development . These mice show reduced frequencies of CD3+ T cells, altered CD4+/CD8+ T cell ratios, and impaired cytolytic activity .

• Embryonic development: Homozygous knockout alleles of RABL3 are embryonic lethal in mice, indicating its crucial role in development .

• KRAS signaling: RABL3 is required for KRAS signaling regulation and modulation of cell proliferation. It functions as a regulator of KRAS prenylation and likely affects prenylation of other small GTPases as well .

• Primary ciliogenesis: RABL3 interacts with RAB11 to regulate ciliary vesicle (CV) formation during early ciliogenesis. Knockout of RABL3 in human RPE1 cells results in significantly fewer primary cilia formation .

Understanding RABL3's functions helps elucidate fundamental biological processes and disease mechanisms, particularly in immune system development and function.

What types of RABL3 antibodies are available for research applications?

Several types of RABL3 antibodies are available for research applications:

• Rabbit Polyclonal antibodies: These recognize multiple epitopes on RABL3 and are suitable for applications including western blot, immunohistochemistry, immunofluorescence, and immunoprecipitation .

• Rabbit Recombinant Monoclonal antibodies: These offer higher specificity and reproducibility compared to polyclonal antibodies. Some are available conjugated to alkaline phosphatase for enhanced detection .

• Mouse Monoclonal antibodies: These provide high specificity for a single epitope and are particularly useful for western blot and immunofluorescence applications with reported dilution ranges of 1:5000-1:50000 for WB and 1:400-1:1600 for IF/ICC .

The choice of antibody depends on your specific application, with monoclonal antibodies generally providing higher specificity while polyclonal antibodies may offer greater sensitivity by recognizing multiple epitopes.

What are the optimal experimental conditions for using RABL3 antibodies in immunohistochemistry?

For optimal immunohistochemistry (IHC) results with RABL3 antibodies:

• Fixation: Formalin-fixed paraffin-embedded (FFPE) tissue sections are commonly used .

• Antigen retrieval: Heat-mediated antigen retrieval with citrate buffer (pH 6.0) is effective for many RABL3 antibodies. For some antibodies, TE buffer (pH 9.0) may provide better results .

• Antibody dilution: The optimal dilution varies by antibody and sample type:

  • For polyclonal antibodies, recommended dilutions range from 1:20 to 1:200

  • For monoclonal antibodies, follow manufacturer's recommendations and optimize for your specific tissue

• Detection system: Standard horseradish peroxidase (HRP)-based detection systems work well with RABL3 antibodies.

• Positive control tissues: Human kidney tissue has been validated for RABL3 IHC and can serve as a positive control .

• Blocking: Use appropriate blocking reagents (typically 1-5% BSA or serum) to minimize background staining.

It's advisable to perform a dilution series to determine optimal antibody concentration for your specific tissue samples.

How can I investigate RABL3's role in lymphocyte development using antibody-based techniques?

To investigate RABL3's role in lymphocyte development, consider these antibody-based approaches:

Flow cytometry for lymphocyte population analysis:

• Use RABL3 antibodies in combination with lymphocyte markers (e.g., B220, CD19, IgM, IgD for B cells; CD3ε, CD4, CD8α for T cells; NK1.1 for NK cells) .

• Example panel: "RBC-depleted samples were stained for 1 h at 4°C, in a 100-μL (1:200 dilution) mixture of fluorescence-conjugated antibodies to 15 cell surface markers encompassing the major immune lineages B220 (clone RA3-6B2), CD19 (clone 1D3), IgM (clone R6-60.2), IgD (clone 11-26c.2a), CD3ε (clone 145-2C11), CD4 (clone RM4-5), CD8α (clone 53-6.7), CD11b (clone M1/70), CD11c (clone HL3), F4/80 (clone BM8.1), CD44 (clone 1M7), CD62L (clone MEL-14), CD5 (clone 53-7.3), CD43 (clone S7), NK1.1 (clone PK136)" .

Immunophenotyping of lymphocyte progenitors:

• Examine bone marrow populations including common lymphoid progenitors (CLPs) and lymphoid-primed multipotent progenitors (LMPPs) using flow cytometry in combination with RABL3 expression analysis .

• Reduced numbers of CLPs and LMPPs were observed in RABL3-mutant mice, indicating its early role in lymphocyte development .

Functional assays with quantitative RABL3 detection:

• Combine cytotoxicity assays for T cells and NK cells with RABL3 expression analysis.

• RABL3-mutant mice show impaired cytolytic activity and defective responses to viral infections (e.g., MCMV) .

Immunoprecipitation strategies:

• Use 0.5-4.0 μg of RABL3 antibody for 1.0-3.0 mg of total protein lysate to investigate RABL3's interaction partners in lymphocytes .

• This approach can help identify potential regulatory mechanisms in lymphocyte development.

What approaches can I use to study RABL3's interaction with GPR89 or RAB11?

To investigate RABL3's interactions with GPR89 or RAB11, consider these methodological approaches:

For RABL3-GPR89 interaction:

• Co-immunoprecipitation (Co-IP): RABL3 strongly associates with and stabilizes GPR89. Wild-type RABL3, but not the mutated form (RABL3 xm), effectively co-precipitates with GPR89 .

  • Use anti-RABL3 antibodies for immunoprecipitation followed by western blot detection of GPR89, or vice versa.

  • Include appropriate controls, such as IgG control and RABL3 mutants.

For RABL3-RAB11 interaction:

• Reciprocal immunoprecipitation: Co-express tagged versions (e.g., Flag-tagged RABL3 and GFP-fused RAB11A) and perform anti-Flag or anti-GFP immunoprecipitation .

• Pull-down assays with recombinant proteins: Using purified recombinant RABL3 and RAB11A proteins from bacterial lysates:
  "Performed a pull-down assay using glutathione sepharose with buffer containing Mg2+, and found that GST-RAB11A specifically pulled down RABL3, demonstrating their direct binding" .

• Guanine nucleotide-dependent binding assays: The RABL3-RAB11 interaction is nucleotide-dependent. "Adding a non-hydrolyzable GTP analog GTPγS or GDP substantially abrogated their association" .

• Co-localization studies: Using immunofluorescence, endogenous RABL3 accumulates around the centrosome during quiescence induction, partially overlapping with RAB11 .

Mutational analysis:

• Test the interaction of various RABL3 mutants (e.g., S20N) with RAB11 mutants (e.g., Q70L, S25N).

• The negative form of RABL3 (S20N) preferentially interacts with the active form of RAB11A (Q70L) .

How can I validate the specificity of my RABL3 antibody for experimental applications?

Validating RABL3 antibody specificity is crucial for reliable experimental results. Consider these methodological approaches:

RABL3 knockout/knockdown controls:

• Use CRISPR/Cas9-generated RABL3 knockout cell lines or siRNA knockdown of RABL3 as negative controls.

• In immunofluorescence experiments, RABL3 antibody-detected puncta were "almost completely absent in Rabl3-2 cells," confirming antibody specificity .

Recombinant protein controls:

• Use purified recombinant RABL3 protein as a positive control in western blot.

• The observed molecular weight should match the calculated size of 26 kDa .

Multiple antibody validation:

• Use multiple antibodies targeting different epitopes of RABL3.

• Consistent results across different antibodies increase confidence in specificity.

Peptide competition assay:

• Pre-incubate the antibody with excess immunizing peptide before application to the sample.

• Specific signal should be significantly reduced or eliminated.

Cross-reactivity testing:

• Test reactivity against related proteins in the Rab subfamily.

• Some antibodies have been "specificity verified on a Protein Array containing target protein plus 383 other non-specific proteins" .

Rescue experiments:

• In functional studies, ectopic expression of RABL3 should rescue phenotypes in knockout/knockdown models.

• "Ectopic expression of RABL3 significantly restored primary cilia in Rabl3-1 and Rabl3-2 cells" .

What methodological considerations are important when using RABL3 antibodies for studying its role in primary ciliogenesis?

When investigating RABL3's role in primary ciliogenesis with antibody-based techniques:

Cell model selection and preparation:

• Retinal pigment epithelial (RPE1) cells are an established model for ciliogenesis studies.

• Induce ciliogenesis by serum starvation: "WT RPE1 cells assembled primary cilia when induced to quiescence by depriving the serum in the culture medium" .

Co-immunofluorescence strategy:

• Use specific markers to visualize primary cilia alongside RABL3:

  • "Primary cilia were visualized by immunofluorescence experiments with two specific antibodies against glutamylated tubulin (Glu. Tub.) and ARL13B" .

  • Include centrosomal markers (e.g., CEP164 for mother centriole) to examine RABL3 localization during early ciliogenesis.

Temporal analysis considerations:

• Examine RABL3 localization at different timepoints during ciliogenesis.

• "Endogenous RABL3 incrementally accumulated in the vicinity of the centrosome during induction to quiescence for 6 h in RPE1 cells" .

Co-localization with RAB11:

• RABL3-positive puncta around the mother centriole partially overlap with RAB11.

• This co-localization provides insight into RABL3's function during early ciliogenesis .

Rescue experiment design:

• In RABL3-depleted cells, reintroduce wild-type or mutant RABL3 to assess functional rescue of ciliogenesis.

• Quantify primary cilia formation in each condition using the cilia markers mentioned above .

Interaction studies with ciliary components:

• Investigate RABL3's interaction with other ciliary proteins using co-immunoprecipitation.

• Focus on proteins involved in ciliary vesicle formation, given RABL3's role in this process .

How can I investigate the structural characteristics of RABL3 in relation to its function?

To investigate RABL3's structural characteristics and relate them to function:

Protein expression and purification:

• Express RABL3 in bacterial systems (e.g., Rosetta DE3) using appropriate vectors (e.g., pHis-parallel).

• For crystallography studies, use truncated RABL3 (aa 2-216) lacking the C-terminus .

• Purification protocol: "The proteins were purified from the clarified lysate with an Ni2+-Sepharose fast flow gravity affinity column at 4°C, followed by ion exchange purification (HiTrap Q HP 5 mL). The peak fractions were collected and incubated with TEV protease overnight at 4°C to remove 6×His tag" .

Site-directed mutagenesis:

• Generate specific RABL3 mutants to study structure-function relationships:

  • The xiamen deletion mutation (Δ43–46) removes four amino acids from the interswitch region .

  • Point mutations like D44G can be used for comparative analysis .

  • Follow standard site-directed mutagenesis protocols: "generated by standard site-directed mutagenesis following the QuikChange II site-directed mutagenesis protocol" .

Biophysical characterization:

• Examine RABL3 dimerization using size exclusion chromatography (e.g., HiLoad 16/600 Superdex 75 gel filtration column) .

• Analyze how mutations affect dimerization and protein conformation.

Structural-functional correlations:

• Relate structural insights to functional outcomes:

  • "RABL3 xm/xm displayed a large compensatory alteration in switch I, which adopted a β-strand configuration normally provided by the deleted interswitch residues, thereby permitting homodimer formation" .

  • "Dysregulated effector binding due to conformational changes in the switch I–interswitch–switch II module likely underlies the xm phenotype" .

Protein-protein interaction analysis:

• Investigate how RABL3 structure influences interactions with partners like GPR89 and RAB11.

• "RABL3, but not RABL3 xm, strongly associated with and stabilized GPR89" .

What techniques can I use to study RABL3's GTPase activity and how does it relate to its cellular functions?

To investigate RABL3's GTPase activity and relate it to cellular functions:

GTPase activity assays:

• Use GTP hydrolysis assays to measure the intrinsic GTPase activity of purified RABL3.

• Compare wild-type RABL3 with mutants affecting GTP binding or hydrolysis.

Nucleotide binding studies:

• Examine binding of GTP, GDP, or non-hydrolyzable analogs (e.g., GTPγS) to RABL3.

• The interaction between RABL3 and RAB11 is nucleotide-dependent: "Adding a non-hydrolyzable GTP analog GTPγS or GDP substantially abrogated their association" .

Mutational analysis approach:

• Generate mutations in RABL3's GTP-binding pocket to create constitutively active (GTP-bound) or inactive (GDP-bound) forms.

• The RABL3 S20N mutant represents a negative (GDP-bound) form that preferentially interacts with active RAB11A (Q70L) .

Cellular localization in relation to nucleotide state:

• Use immunofluorescence to examine how different nucleotide-bound states affect RABL3's subcellular localization.

• Correlate localization with function in processes like ciliogenesis or lymphocyte development.

Functional readouts:

• In lymphocyte development: Analyze how RABL3's GTPase activity affects B cell, T cell, and NK cell development and function .

• In ciliogenesis: Examine how different nucleotide-bound states affect RABL3's role in ciliary vesicle formation .

• In KRAS signaling: Investigate how RABL3's GTPase activity influences KRAS prenylation and downstream signaling .

What are the most common challenges when using RABL3 antibodies, and how can I overcome them?

Common challenges with RABL3 antibodies and their solutions include:

Poor signal intensity:

• Solution: Optimize antibody concentration by testing a range of dilutions. Recommended dilutions vary by application:

  • Western blot: 1:1000-1:50000 depending on the antibody

  • IHC: 1:20-1:200

  • IF/ICC: 1:200-1:1600

• Solution: Enhance signal using more sensitive detection systems or longer exposure times.

• Solution: Improve antigen retrieval methods for IHC (try both citrate buffer pH 6.0 and TE buffer pH 9.0) .

High background:

• Solution: Increase blocking time and concentration (e.g., 5% BSA or normal serum).

• Solution: Optimize antibody dilution to reduce non-specific binding.

• Solution: Include additional washing steps with higher salt concentration.

• Solution: For IF/ICC, include a permeabilization optimization step.

Inconsistent results:

• Solution: Standardize sample preparation procedures.

• Solution: Use positive control samples (e.g., human kidney tissue for IHC; HEK-293, U-251, or U2OS cells for WB) .

• Solution: Ensure proper storage of antibodies: "Store at -20°C. Stable for one year after shipment" .

Cross-reactivity:

• Solution: Use monoclonal antibodies when higher specificity is required.

• Solution: Validate antibody specificity using RABL3 knockout or knockdown samples.

• Solution: Consider pre-adsorption of the antibody with recombinant RABL3 protein as a control.

Epitope masking in fixed samples:

• Solution: Optimize fixation time and conditions.

• Solution: Try different antigen retrieval methods for IHC or IF on fixed samples.

• Solution: Consider using multiple antibodies targeting different epitopes of RABL3.

How can I optimize the detection of low-abundance RABL3 in specific cell types or tissues?

For detecting low-abundance RABL3 in specific cells or tissues:

Sample preparation optimization:

• Enrich the target cell population using cell sorting or isolation techniques before analysis.

• For tissue samples, use laser capture microdissection to isolate specific regions of interest.

Signal amplification strategies:

• Use tyramide signal amplification (TSA) for immunohistochemistry or immunofluorescence.

• Consider biotin-streptavidin amplification systems.

• For western blot, use highly sensitive chemiluminescent substrates or near-infrared fluorescent detection.

Protein concentration techniques:

• For western blot, increase the amount of protein loaded per lane.

• Use immunoprecipitation to concentrate RABL3 before detection: "Use 0.5-4.0 ug of antibody for 1.0-3.0 mg of total protein lysate" .

Alternative detection methods:

• Consider mass spectrometry-based approaches for very low abundance detection.

• Use proximity ligation assay (PLA) to detect RABL3 interactions with high sensitivity.

• For protein interactions, antibody-array interaction mapping (AAIM) can detect and measure interactions among a defined set of proteins with high sensitivity .

Increase antibody specificity and sensitivity:

• Use monoclonal antibodies for higher specificity.

• Optimize antibody concentration and incubation conditions (time, temperature).

• For WB, try longer exposure times or more sensitive detection methods.

Controls and validation:

• Include positive controls where RABL3 is known to be expressed.

• Use RABL3-overexpressing cells as standard for assay optimization.

• Include negative controls (RABL3 knockout/knockdown) to confirm signal specificity.

How should I interpret variations in RABL3 expression between different cell types or experimental conditions?

When interpreting variations in RABL3 expression:

Baseline expression considerations:

• Establish normal RABL3 expression levels in your cell types of interest using validated antibodies.

• Consider tissue-specific expression patterns when interpreting results.

Quantification methods:

• For western blot: Use densitometry with appropriate normalization to housekeeping proteins.

• For IHC/IF: Quantify signal intensity, percentage of positive cells, or subcellular localization patterns.

• For flow cytometry: Analyze mean fluorescence intensity and percentage of positive cells.

Statistical analysis approach:

• Apply appropriate statistical tests based on your experimental design.

• Include sufficient biological and technical replicates for robust analysis.

• Present data with proper measures of central tendency and dispersion.

Functional correlations:

• Relate RABL3 expression changes to functional outcomes:

  • In lymphocytes: Correlate with cell development, activation status, or effector functions .

  • In primary cilia: Relate to ciliogenesis efficiency and ciliary vesicle formation .

  • In KRAS signaling: Connect to cell proliferation and oncogenic pathways .

Confounding factors to consider:

• Cell cycle stage may affect RABL3 expression or localization.

• Stress conditions might alter RABL3 levels or activity.

• Consider post-translational modifications that might affect antibody recognition.

Contextual interpretation:

• Interpret RABL3 expression in the context of related proteins (e.g., RAB11, GPR89).

• Consider the nucleotide-bound state of RABL3, which may affect its function and interactions.

What experimental approaches can help differentiate between direct and indirect effects of RABL3 in cellular pathways?

To differentiate between direct and indirect effects of RABL3:

Temporal analysis:

• Perform time-course experiments after RABL3 manipulation to distinguish immediate (likely direct) from delayed (possibly indirect) effects.

• Use inducible expression or degradation systems for precise temporal control.

Genetic rescue strategies:

• Complement RABL3 knockout/knockdown with:

  • Wild-type RABL3 (should rescue direct effects)

  • Specific RABL3 mutants (e.g., GTPase-deficient mutants or interaction-deficient mutants)

  • Related Rab family proteins to test functional redundancy

Direct binding assays:

• Use purified recombinant proteins to test direct interactions:

  • Pull-down assays: "Performed a pull-down assay using glutathione sepharose with buffer containing Mg2+, and found that GST-RAB11A specifically pulled down RABL3, demonstrating their direct binding" .

  • Surface plasmon resonance (SPR) or microscale thermophoresis (MST) for quantitative binding analysis.

Proximity-based interaction detection:

• BioID or APEX2 proximity labeling to identify proteins in close proximity to RABL3 in living cells.

• Förster resonance energy transfer (FRET) or bimolecular fluorescence complementation (BiFC) to visualize direct interactions in cells.

Pathway dissection approaches:

• Use specific inhibitors of known RABL3-dependent pathways to block potential indirect effects.

• In lymphocyte development: Analyze specific stages of development to identify the earliest RABL3-dependent processes .

• In ciliogenesis: Focus on early events like ciliary vesicle formation where RABL3 directly interacts with RAB11 .

Domain-specific manipulations:

• Design mutations affecting specific RABL3 interaction surfaces or functions.

• The xiamen mutation (Δ43–46) specifically disrupts the interswitch region, affecting RABL3's interaction with GPR89 .

By combining these approaches, researchers can build a comprehensive understanding of direct RABL3 functions versus downstream indirect effects.