ARL2BP Antibody

What is ARL2BP and what functions does it serve in cellular biology?

ARL2BP is a 19 kDa protein (observed at approximately 20-21 kDa on Western blots) that functions as an effector of small GTPases ARL2 and ARL3. Together with ARL2, it plays a significant role in the nuclear translocation, retention, and transcriptional activity of STAT3 . The protein is also known by several alternative names including BART, BART1, ADP-ribosylation factor-like protein 2-binding protein, and Binder of ARF2 protein 1 . ARL2BP has been identified as a centrosome-associated protein in various mammalian cell lines and has critical functions in ciliary biology. Mutations in the ARL2BP gene are causative for autosomal-recessive retinitis pigmentosa (RP66), highlighting its importance in retinal function and maintenance . The protein's role in primary cilia function has been demonstrated through experimental depletion, which causes cilia shortening, suggesting it is essential for normal photoreceptor maintenance and function .

Where is ARL2BP localized in photoreceptor cells and what does this suggest about its function?

Immunofluorescence and immunoelectron microscopy studies in mouse retina have revealed specific subcellular localization patterns for ARL2BP. The protein localizes to several critical structures in photoreceptors:

• The basal body of the connecting cilium

• The cilium-associated centriole

• The ciliary rootlet

• The distal connecting cilia

• The periciliary extension of the inner segment

This localization pattern is particularly significant as these structures serve as important regulation points for protein trafficking into the primary cilium. The basal body complex and periciliary extension function as docking sites for pericentriolar transport vesicles and intraflagellar transport (IFT) particles . ARL2BP's presence in distal connecting cilia, where new outer-segment disks are formed, suggests a potential role in disk neogenesis and indicates a close relationship with IFT molecules that show similar spatial distribution in ciliary subcompartments . This localization pattern strongly suggests ARL2BP functions in the targeting, docking, or loading of proteins and/or vesicles in the periciliary region for cilia-associated traffic.

How does the interaction between ARL2BP and ARL2 influence ciliary localization?

The functional significance of this interaction is further supported by studies of the p.Met45Arg mutation in ARL2BP, which causes retinitis pigmentosa. This amino acid substitution reduces binding to ARL2 and results in loss of ARL2BP localization at the basal body in ciliated nasal epithelial cells . The mutation does not affect protein stability or expression levels but specifically disrupts the ARL2-ARL2BP interaction. These findings collectively demonstrate that the precise molecular interaction between ARL2 and ARL2BP is essential for proper localization and, consequently, normal ciliary function.

What validation methods should I use to confirm ARL2BP antibody specificity?

Proper validation of ARL2BP antibodies is crucial for ensuring experimental reliability. Based on the search results, several complementary approaches are recommended:

• Knockout/Knockdown validation: The gold standard validation approach involves using knockout cell lines or knockdown experiments. For example, ab188322 Anti-ARL2BP antibody was validated using the Human ARL2BP knockout HeLa cell line (ab265269), demonstrating loss of signal in Western blot when the target protein was absent . Similarly, siRNA knockdown of ARL2BP showed reduction of signal in immunocytochemistry and Western blot assays .

• Multiple antibody comparison: Using at least two different antibodies against ARL2BP that recognize different epitopes can confirm specificity of localization signals. This approach was employed in mouse retina studies where localization at the distal connecting cilia was corroborated with a second ARL2BP antibody .

• Cross-species reactivity testing: Testing the antibody across multiple species samples (human, mouse, rat) can help establish conservation of epitope recognition and specificity .

• Multiple application testing: Validating the antibody across different applications (WB, ICC/IF, IHC, IP, Flow cytometry) helps establish versatility and consistent target recognition .

• Band size verification: Confirming that the observed band size (20-21 kDa) matches the predicted molecular weight (19 kDa) with allowance for post-translational modifications .

What controls should I include when using ARL2BP antibodies in my experiments?

Robust experimental design requires appropriate controls when using ARL2BP antibodies:

For co-localization studies, include appropriate markers for subcellular structures. For instance, when studying ARL2BP at the basal body, use established markers like centrin-3 (for cilia, basal body, and centriole) and PCM-1 (for periciliary regions) .

What are the optimal protocols for Western blot detection of ARL2BP?

Based on the validated protocols from the search results, the following recommendations can be made for Western blot detection of ARL2BP:

Sample Preparation and Loading:

• Use 10-20 μg of total protein lysate per lane

• Tested cell lines with reliable ARL2BP expression include HeLa, HUVEC, MCF7, A549, and HepG2

• Tissue samples with confirmed expression include human fetal brain, mouse brain, mouse heart, and rat spleen

Antibody Dilutions and Incubation:

• For ab188322 (rabbit monoclonal): Use 1/1000 to 1/10000 dilution depending on sample type

• For 10090-2-AP (rabbit polyclonal): Use 1/200 to 1/1000 dilution

• Incubate primary antibody overnight at 4°C

• Secondary antibody recommendations:

  • Goat anti-Rabbit IgG H&L (IRDye® 800CW) at 1/10000-1/20000 dilution

  • Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugate at 1/1000 dilution

Expected Results:

• Predicted band size: 19 kDa

• Observed band size: 19-21 kDa (slight variation may occur between species and cell types)

For optimal results, include positive controls (known ARL2BP-expressing samples) and negative controls (ARL2BP knockout samples if available) to confirm antibody specificity.

What is the recommended protocol for immunohistochemistry with ARL2BP antibodies?

Successful immunohistochemistry (IHC) for ARL2BP requires careful attention to antigen retrieval and antibody dilution. Based on validated protocols:

Tissue Preparation:

• Use paraffin-embedded tissue sections

• Successfully tested tissues include human kidney, rat liver, and human pancreas cancer tissue

Antigen Retrieval:

• Perform heat-mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol

• Alternative method: citrate buffer pH 6.0 may also work for some tissue types

Antibody Dilutions:

• For ab188322: Use at 1/50 dilution

• For 10090-2-AP: Use at 1/250-1/1000 dilution

• Secondary detection: prediluted HRP Polymer for Rabbit IgG

• Counterstain with Hematoxylin for nuclear visualization

Controls:

• Include a negative control section (omitting primary antibody)

• If possible, include tissues from knockout models as specificity controls

The search results show successful staining in kidney tubules and liver tissue with clearly defined subcellular localization patterns. When optimizing the protocol for new tissue types, it is advisable to test a range of antibody dilutions and both antigen retrieval methods.

How should I prepare samples for immunofluorescence detection of ARL2BP in ciliated cells?

Immunofluorescence detection of ARL2BP, particularly in ciliated cells where it localizes to specific subcellular structures, requires careful sample preparation:

Cell Fixation:

• Fix cells with 4% paraformaldehyde to preserve ciliary structures

• For intracellular staining (as in flow cytometry), permeabilize with 90% methanol

• For ICC/IF applications, use 0.1-0.5% Triton X-100 for permeabilization after fixation

Antibody Dilutions:

• For ab188322: Use at 1/100 dilution for immunofluorescence

• For visualization, use fluorophore-conjugated secondary antibodies such as Goat anti-rabbit IgG (Alexa Fluor® 488) at 1/200-1/2000 dilution

Co-staining Recommendations:

• Include ciliary markers for co-localization studies:

  • Centrin-3 for basal body, centriole, and cilium identification

  • PCM-1 for periciliary region

  • ARL13B for ciliary membrane labeling

• Counterstain nuclei with DAPI for proper cellular orientation

Sample Types:

• Successfully tested cell lines include A549, NIH 3T3, ARPE19, and SK-N-SH neuroblastoma cells

• For ciliary studies, ensure cells are grown to confluence and serum-starved for 24-48 hours to induce ciliation

For optimal visualization of ARL2BP at the basal body and in ciliary compartments, high-resolution confocal microscopy is recommended, with Z-stack acquisition to capture the three-dimensional structure of the cilium.

Why might I observe different molecular weights for ARL2BP in Western blots?

Researchers often observe variability in the apparent molecular weight of ARL2BP in Western blots. While the calculated molecular weight is 19 kDa, the observed molecular weight is typically reported as 20-21 kDa . Several factors could explain these discrepancies:

• Post-translational modifications: Phosphorylation, glycosylation, or other modifications can alter protein migration in SDS-PAGE.

• Species differences: Slight variations in molecular weight may be observed between human, mouse, and rat ARL2BP due to species-specific modifications or processing.

• Technical factors: Running conditions, gel percentage, and buffer systems can all influence apparent molecular weight.

• Sample preparation: Different lysis buffers or denaturation conditions might affect protein conformation and SDS binding, altering migration.

When interpreting Western blot results for ARL2BP, it's important to note that both the 19 kDa and 20-21 kDa bands have been validated as specific in control experiments using knockout cell lines . The specificity of the observed band can be confirmed by using ARL2BP knockout cell lysates (such as Human ARL2BP knockout HeLa cell lysate ab258312) as negative controls .

What are common pitfalls in interpreting ARL2BP localization data?

Interpreting ARL2BP localization data requires careful consideration of several potential pitfalls:

• Overlapping subcellular structures: The basal body, adjacent centriole, and ciliary rootlet are in close proximity, making it challenging to distinguish between these structures without super-resolution microscopy or co-localization with specific markers. Using markers like centrin-3 and PCM-1 in co-staining experiments can help resolve these structures .

• Fixation artifacts: Different fixation methods can alter the appearance of ciliary structures. Paraformaldehyde fixation is generally recommended for maintaining ciliary architecture, but methanol fixation may better preserve some centrosomal epitopes .

• Antibody specificity concerns: ARL2BP shows both cytoplasmic and basal body localization. Non-specific antibodies may give misleading results. Validation using siRNA knockdown of ARL2BP is crucial to confirm specificity of the observed signals .

• Developmental and cell cycle variations: ARL2BP localization may vary depending on cell cycle stage or developmental status of the cilium. In non-ciliated cells or cells with resorbed cilia, interpretation of localization can be misleading.

• Mutant protein localization: When studying ARL2BP mutants (like p.Met45Arg), the mutation may affect antibody recognition or protein localization. Using epitope-tagged constructs in parallel can help distinguish between these possibilities .

To avoid these pitfalls, always include appropriate controls, use multiple antibodies when possible, and corroborate immunofluorescence findings with biochemical approaches such as fractionation or proximity labeling.

How can I design experiments to study the interaction between ARL2BP and ARL2 in vivo?

The interaction between ARL2BP and ARL2 is critical for proper ciliary function, as demonstrated by the finding that depletion of ARL2 causes displacement of ARL2BP from the basal body . To study this interaction in vivo, consider the following experimental approaches:

• Proximity Ligation Assay (PLA): This technique can detect protein-protein interactions with high specificity and sensitivity in fixed cells or tissues. Use validated antibodies against ARL2BP and ARL2 that work in immunofluorescence.

• FRET/FLIM Analysis: Express fluorescently tagged ARL2BP and ARL2 in ciliated cells and measure Förster resonance energy transfer to assess direct interactions in living cells.

• Co-immunoprecipitation with Mutation Analysis: Compare co-IP efficiency between wild-type ARL2BP and mutant versions (such as p.Met45Arg) to identify critical residues for interaction. For example, research has shown that the p.Met45Arg substitution reduced binding to ARL2 . Design a panel of mutations to map the interaction interface.

• Protein Complementation Assays: Split fluorescent protein approaches (BiFC) or split luciferase assays can provide direct visualization of the ARL2BP-ARL2 interaction in living cells.

• Structure-Function Analysis: Based on immunoprecipitation studies with wild-type and mutant proteins (like the p.Met45Arg substitution which showed reduced ARL2 binding), design a series of domain deletions or point mutations to map the interaction interface .

• GTPase State-Dependent Binding Assays: Since ARL2 is a small GTPase, compare binding of ARL2BP to wild-type ARL2, constitutively active (GTP-bound) mutants, and dominant-negative (GDP-bound) mutants to determine if the interaction is nucleotide-dependent.

These approaches can be combined with ciliary function assays (such as measuring cilia length) to correlate biochemical interactions with functional outcomes.

What approaches can be used to investigate the role of ARL2BP in ciliary trafficking?

ARL2BP localizes to key structures involved in ciliary protein trafficking, including the basal body and periciliary extension of the inner segment . To investigate its role in ciliary trafficking:

• Live Cell Imaging of Cargo Transport: Express fluorescently tagged ciliary cargoes (e.g., rhodopsin in photoreceptors) and monitor their transport dynamics in cells with normal or depleted ARL2BP. Use FRAP (Fluorescence Recovery After Photobleaching) to measure transport rates.

• Ultrastructural Analysis: Employ transmission electron microscopy and immunoelectron microscopy to examine the ultrastructure of the ciliary pocket, transition zone, and outer segment disk formation in tissues with ARL2BP mutations or knockdown .

• Interactome Analysis: Use BioID, APEX proximity labeling, or quantitative mass spectrometry to identify ARL2BP-interacting proteins at the basal body and compare with known components of ciliary trafficking pathways.

• Ciliary Protein Accumulation Assays: Measure the accumulation of various ciliary proteins in the presence or absence of ARL2BP to determine if specific cargo subsets are affected.

• Rescue Experiments with Trafficking Mutants: Design ARL2BP mutants that disrupt specific protein-protein interactions and test their ability to rescue ciliary defects in ARL2BP-depleted cells.

• Functional Assessment of IFT: Based on the observation that ARL2BP shows similar spatial distribution to IFT molecules in ciliary subcompartments , analyze whether ARL2BP depletion affects IFT train movement using live imaging of tagged IFT proteins.

• Analysis of Disk Morphogenesis: Since ARL2BP is present in distal connecting cilia where new outer-segment disks are formed, investigate its role in disk neogenesis using high-resolution microscopy and membrane trafficking assays .

These approaches can help determine whether ARL2BP functions in specific aspects of ciliary protein trafficking, such as cargo selection, docking of transport vesicles, or regulation of IFT-mediated transport.

How can I assess the functional consequences of ARL2BP mutations on cilia formation and function?

To assess the functional consequences of ARL2BP mutations (such as those causing retinitis pigmentosa), a multi-tiered approach is recommended:

• Cilia Length and Morphology Analysis: Measure cilia length and morphology in cells expressing wild-type or mutant ARL2BP. Research has shown that depletion of ARL2BP causes significant reduction in cilia length in ARPE19 cells . Compare multiple cell types to assess tissue-specific effects.

• Ciliary Protein Localization: Examine the localization of key ciliary proteins in cells expressing wild-type or mutant ARL2BP to determine if trafficking defects are general or cargo-specific.

• Ciliary Function Assays:

  • For sensory cilia: Measure calcium influx in response to appropriate stimuli

  • For motile cilia: Assess ciliary beat frequency and waveform

  • For photoreceptors: Test light responses using electroretinography

• CRISPR/Cas9 Knock-in Models: Generate cell lines or animal models with specific ARL2BP mutations (such as the c.134T>G (p.Met45Arg) mutation identified in retinitis pigmentosa patients) . Compare phenotypes across different mutations.

• Protein-Protein Interaction Analysis: Compare interaction profiles of wild-type and mutant ARL2BP with known partners like ARL2. For example, the p.Met45Arg substitution reduced binding to ARL2 and caused loss of ARL2BP localization at the basal body in ciliated cells .

• Rescue Experiments: Test whether wild-type ARL2BP can rescue defects in cells or organisms lacking endogenous ARL2BP, and compare with the rescue capability of disease-associated mutants.

• Temporal Protein Dynamics: Using live-cell imaging with fluorescently tagged proteins, compare the dynamics of wild-type and mutant ARL2BP during ciliogenesis, maintenance, and resorption.

This comprehensive approach can provide insights into both the molecular mechanisms disrupted by ARL2BP mutations and their functional consequences at the cellular and organismal levels.