ARL13B Antibody

What is ARL13B and why is it important for ciliary research?

ARL13B (ADP-ribosylation factor-like protein 13B), also known as ARL2L1, is a small ciliary G protein of the Ras superfamily primarily localized in cilia. It plays critical roles in cilium biogenesis and Sonic hedgehog (Shh) signaling, making it essential for developmental processes and cellular homeostasis. The importance of ARL13B stems from its involvement in multiple biological contexts:

ARL13B functions as a key regulator of ciliary structure and signaling pathways. It is required for proper cilia formation, and mutations in ARL13B cause Joubert syndrome (JS), an autosomal recessive disorder characterized by distinctive cerebellar malformation. The protein contains both N-terminal and C-terminal guanine nucleotide-binding motifs that mediate its GTPase activity .

Most significantly for researchers, ARL13B antibodies serve as excellent markers for primary cilia across diverse cell types and tissues, enabling detailed studies of ciliary biology, development, and disease mechanisms .

What applications can ARL13B antibody be used for in experimental research?

ARL13B antibodies have been validated for multiple experimental applications, providing researchers with versatile tools for studying ciliary biology:

The extensive validation across multiple applications makes ARL13B antibodies particularly reliable tools for ciliary research. The high number of publications using IF applications (730+) reflects its widespread adoption as a standard marker for cilia visualization in developmental and cell biological studies .

How should I optimize fixation methods for ARL13B immunofluorescence staining?

Successful immunofluorescence detection of ARL13B requires careful consideration of fixation protocols, as different methods can significantly impact antibody performance:

Paraformaldehyde (PFA) Fixation:

• 4% PFA for 10-15 minutes at room temperature

• Commonly used and works well in many cell types

• Requires permeabilization with 0.1% Triton X-100 + 0.1% Tween + 0.01% SDS in PBS for optimal results

• Validated in hTERT-RPE1, NIH3T3, and MDCK cells

Methanol Fixation:

• Cold methanol for 10 minutes at -20°C

• Often provides superior ciliary staining compared to PFA

• Follow with rehydration using PBS for 5 minutes

• Particularly effective for ciliary axoneme visualization

• Works well in MDCK cells as reported by multiple researchers

Researcher feedback indicates both methods can yield excellent results, though methanol fixation often provides better signal-to-noise ratio for ciliary ARL13B. A verified protocol from customer testimonials specifies: "Cells were fixed in cold methanol for 10 minutes at -20°C, rehydrated with PBS for 5 minutes, and permeabilized with 0.1% Triton + 0.1% Tween + 0.01% SDS in PBS for 5 minutes. Primary antibody was diluted in blocking buffer (5% BSA + 0.1% Tween in PBS) at 1:200 for 1 hour at room temperature" .

What is the significance of multiple molecular weight bands observed in ARL13B Western blots?

ARL13B antibodies typically detect two distinct bands in Western blot analysis, which have important biological significance:

Expected vs. Observed Molecular Weights:

• Calculated molecular weight: 48 kDa (based on amino acid sequence)

• Observed lower band: 40-48 kDa (corresponds to unmodified ARL13B)

• Observed upper band: 60-66 kDa (represents post-translationally modified form)

The 60-66 kDa band likely represents ARL13B with post-translational modifications, though the exact nature of these modifications has not been fully characterized. Both bands are specific to ARL13B, as verified through knockout validation studies. The specificity of these bands has been confirmed using multiple approaches including knockout/knockdown validation and peptide competition assays .

For optimal Western blot analysis, researchers should:

• Use 10% acrylamide gels to provide good separation of both bands

• Include phosphatase/protease inhibitors in lysis buffers to preserve modified forms

• Monitor both bands for complete analysis of ARL13B expression

• Consider the ratio between modified and unmodified forms as potentially biologically relevant

Which species does the ARL13B antibody react with in experimental systems?

ARL13B antibodies demonstrate reactivity across multiple species, making them versatile tools for comparative studies:

The broad cross-reactivity of ARL13B antibodies makes them particularly valuable for evolutionary and comparative studies of ciliary biology. For species with predicted rather than confirmed reactivity, validation experiments should be conducted prior to extensive use .

Customer testimonials indicate successful use in diverse species: "This antibody works really well in human and mouse cell lines and in zebrafish samples" and "Very specific antibody for primary cilia. We use it in chicken embryos spinal cord" .

How does ARL13B localization affect experimental interpretation in ciliary and signaling studies?

The subcellular localization of ARL13B has profound implications for experimental design and data interpretation in ciliary biology research:

ARL13B shows distinct subcellular localization patterns:

• Primary Cilium: Highly enriched and traditionally considered its primary functional site

• Cytoplasm: Detectable levels with appropriate exposure/sensitivity

• Other Membrane Compartments: Potential association with vesicular structures

Research with the ARL13B V358A mutant has revealed that ARL13B can regulate Sonic hedgehog signaling from outside primary cilia. This mutant disrupts ciliary localization but maintains signaling function, indicating ARL13B has separable roles in cilia structure and signaling:

This discovery necessitates careful experimental design when studying ARL13B function:

• Detection of non-ciliary ARL13B may require over-exposure techniques

• Quantitative approaches (cilium:cell body ratio measurements) provide more objective assessment

• Phenotypes previously attributed to ciliary defects may need reevaluation

• Co-localization studies should include appropriate controls and quantification

When interpreting ARL13B localization data, researchers should consider that phenotypes may result from disrupted functions in specific subcellular compartments rather than assuming ciliary defects explain all observed outcomes.

What controls should be included when using ARL13B antibody in studies of ciliopathies?

Comprehensive controls are essential for rigorous ciliopathy research using ARL13B antibodies:

Essential Antibody Validation Controls:

• Knockout/Knockdown Controls: Use Arl13b knockout cells/tissues (e.g., Arl13b^hnn/hnn) to verify antibody specificity

• Peptide Competition: Pre-incubate antibody with immunizing peptide to confirm epitope specificity

• Secondary-only Controls: Omit primary antibody to assess background from secondary antibody

• Isotype Controls: Use non-specific IgG from same host species to evaluate non-specific binding

Critical Biological Controls for Ciliopathy Studies:

• Known Ciliopathy Models: Include established mutants (e.g., Ift88, Kif3a, Cep290) for phenotypic benchmarking

• Tissue-Specific Controls: Use adjacent normal tissue in patient samples to account for tissue variability

• Developmental Controls: Include age-matched samples for developmental studies

• Rescue Experiments: Re-express wild-type protein in mutant background to confirm causality

Research with ARL13B V358A mutants demonstrates effective control implementation:

• Researchers used Arl13b^hnn/hnn MEFs as negative controls for antibody specificity

• They determined background staining levels through quantitative ratio measurements

• Extended analysis with multiple over-exposure settings captured different abundance levels

• Additional ciliary markers (IFT88) provided co-localization confirmation

How can ARL13B antibody be combined with other markers for comprehensive cilia studies?

Strategic combinations of ARL13B with other ciliary markers enable sophisticated analysis of ciliary structure, function, and associated signaling pathways:

Optimized Ciliary Structure Analysis Combinations:

These combinations provide complementary information about ciliary architecture. Customer testimonials support their effectiveness: "NIH-3T3 stained in DAPI (nucleus), FITC (gamma tubulin), Cy3 (Arl13b). On the merged channel, we can conclude that all Cy3 signal is validated by the FITC one (gamma tubulin is present at the base of the primary cilia)" .

Functional Domain and Signaling Combinations:

For optimal co-staining results:

• Select primary antibodies raised in different host species (e.g., rabbit anti-ARL13B with mouse anti-acetylated α-tubulin)

• Choose spectrally separated fluorophores (e.g., Alexa-488 and Alexa-555/594)

• Balance exposure settings to capture both abundant and less abundant markers

• Consider confocal microscopy for densely ciliated samples

Advanced analytical approaches include quantitative co-localization (Pearson's or Manders' coefficients), linescan analysis to assess protein distribution along ciliary length, and potentially super-resolution microscopy for nanoscale localization studies.

What considerations are important when using ARL13B antibody in studies of Sonic hedgehog signaling?

When utilizing ARL13B antibodies in Sonic hedgehog (Shh) signaling research, several critical factors must be considered:

Key Functional Relationships:

• ARL13B is required for proper Shh signal transduction

• ARL13B mutations alter ciliary localization of Shh pathway components

• ARL13B can regulate Shh signaling from outside primary cilia (as demonstrated with ARL13B V358A)

Essential Experimental Design Considerations:

The discovery that ARL13B regulates cilia length and Shh signaling through distinct mechanisms has significant implications: "Our data support ARL13B regulating different biological processes from its distinct subcellular localizations consistent with how other GTPases act from multiple sites in cells through different effectors. ARL13B V358A disrupts cilia localization of INPP5E and ARL3, but not Shh components" .

This separation of function necessitates careful experimental approach:

• Use domain-specific mutations that alter localization without eliminating expression

• Include both ciliary structural markers and multiple Shh pathway readouts

• Consider that ARL13B may affect component trafficking without altering total expression

• Distinguish between direct signaling effects and indirect consequences of ciliary alterations

These considerations enable more accurate interpretation of how ARL13B contributes to both ciliary structure and Shh signal transduction.

How can I optimize ARL13B antibody staining for detecting primary cilia in different tissue types?

Optimizing ARL13B staining for primary cilia detection across diverse tissue types requires tissue-specific adjustments and methodological refinements:

Tissue-Specific Optimization Strategies:

For antigen retrieval in immunohistochemical applications, "suggested antigen retrieval with TE buffer pH 9.0; alternatively, antigen retrieval may be performed with citrate buffer pH 6.0" .

Systematic Optimization Approach:

• Antibody Titration:

  • Perform systematic dilution series (1:50, 1:100, 1:200, 1:400, 1:800)

  • Assess signal-to-noise ratio at each dilution

  • Remember optimal dilution may differ between tissue types

• Incubation Parameter Optimization:

  • Compare room temperature (1-2 hours) vs. 4°C (overnight) incubation

  • Test different blocking agents (BSA, normal serum, commercial blockers)

  • Adjust washing stringency (salt concentration, detergent levels)

• Image Acquisition Optimization:

  • Use Z-stack imaging to capture entire ciliary length

  • Apply deconvolution for improved resolution

  • Maintain consistent exposure settings for quantitative analyses

Customer testimonials indicate successful optimization: "I use this antibody for IHC on brain tissue sections and it generally works well" and "Excellent specificity with minimal background/nonspecific binding; This antibody is ideal because it works just as well in embryonic mouse tissue as it does in adult" .

How can I troubleshoot weak or non-specific staining with ARL13B antibody?

Troubleshooting ARL13B antibody staining issues requires systematic evaluation of multiple experimental parameters:

Addressing Weak or Absent Signal:

Resolving High Background/Non-specific Staining:

A step-by-step troubleshooting protocol should begin by validating ciliation status using acetylated α-tubulin, followed by systematic optimization of fixation/permeabilization, antibody dilution, and incubation parameters .

Successful protocols from verified customers include:
"Great antibody for centrosome and cilia visualization in RPE1 cells. Cells were fixed in cold methanol for 10' at -20C, rehydrated with PBS for 5', and permeabilized with 0.1% Triton + 0.1% Tween +0.01% SDS in PBS for 5'. Cells were finally incubated with blocking buffer (5% BSA+ 0.1% Tween in PBS) for 30' at RT. Primary antibody was diluted in blocking buffer 1:200 and incubated for 1h at room temperature" .

What role does ARL13B play in reproductive physiology research?

ARL13B has emerging significance in reproductive biology research, particularly in the study of male reproductive tract development and function:

Recent studies have investigated ARL13B's role in the efferent ductules, which are essential tubules in the male reproductive tract critical for fertility. In conditional knockout (cKO) mouse models, ARL13B depletion disrupts reproductive tract homeostasis:

"In cKO mice, Lcn6 transcript was not only detected in the IS of the epididymis but also in epithelial cells and somatic cells invading the lumen of the efferent ducts. These findings suggest that the depletion of Arl13b disrupts the balance of the immune system, leading to mononuclear phagocytes infiltration into the lumen area, and prompting specific epithelial cells to initiate the expression of various immune-related markers associated with the innate immune response" .

This research demonstrates that ARL13B functions through primary cilia to maintain male reproductive tract physiology through several mechanisms:

• Regulation of immune homeostasis in the efferent ductules

• Influence on epithelial cell gene expression patterns

• Prevention of pathological cell infiltration into the luminal compartment

• Maintenance of proper tissue architecture necessary for fertility

These findings expand our understanding of ARL13B beyond its established roles in brain development and Sonic hedgehog signaling, highlighting its importance in reproductive physiology and potentially offering insights into certain forms of male infertility.

What is the relationship between ARL13B and human disease, particularly ciliopathies?

ARL13B has significant clinical relevance, with mutations directly linked to human ciliopathies:

Joubert Syndrome (JS):
ARL13B mutations are the cause of Joubert syndrome type 8 (JBTS8), an autosomal recessive disorder characterized by a distinctive cerebellar malformation. "Defects in ARL13B are the cause of Joubert syndrome (JS) which is an autosomal recessive disorder characterized by a distinctive cerebellar malformation" .

The connections between ARL13B and ciliopathies stem from its essential roles in:

• Cilium biogenesis and maintenance

• Regulation of sonic hedgehog signaling

• Proper ciliary protein trafficking

• Ciliary membrane composition

Research applications studying ARL13B in disease contexts include:

• Analysis of patient-derived mutations to understand structure-function relationships

• Development of animal models mimicking human ARL13B mutations

• Investigation of cellular mechanisms underlying JS pathology

• Identification of potential therapeutic targets for ciliopathies

For researchers studying ARL13B in disease contexts, it's important to note that ARL13B functions through distinct mechanisms:

• Regulation of cilia length/structure

• Control of signaling pathways (particularly Shh)

• Trafficking of specific ciliary cargo proteins (INPP5E, ARL3)

Understanding these separable functions helps clarify how different mutations might cause distinct disease phenotypes, even within the spectrum of Joubert syndrome. This knowledge is essential for accurate genotype-phenotype correlations and potential therapeutic development for ciliopathies.