ARL2BP Antibody, FITC conjugated

What is ARL2BP and what is its primary biological function?

ARL2BP (ADP-ribosylation factor-like protein 2-binding protein) is a 19 kDa protein that specifically binds to ARL2.GDP with high affinity. Together with ARL2, it plays a crucial role in the nuclear translocation, retention, and transcriptional activity of STAT3 . ARL2BP may also function as an effector of ARL2. This protein is also known by several other names including BART, BART1, ARF-like 2-binding protein, and Binder of ARF2 protein 1 .

Research has established a photoreceptor-specific role for ARL2BP in axoneme length regulation, disk morphogenesis, and ciliary doublet formation . The absence of ARL2BP leads to progressive loss of function and degeneration of photoreceptors, with early morphological defects including abnormal ciliary microtubule doublet structure with open B-tubules .

What is the connection between ARL2BP and retinitis pigmentosa?

ARL2BP has been linked to retinitis pigmentosa (RP), a genetic disorder causing retinal degeneration. Studies utilizing ARL2BP knockout (KO) mouse models have demonstrated that ARL2BP is necessary for photoreceptor ciliary doublet formation and axoneme elongation, processes required for outer segment morphogenesis and vision . The absence of ARL2BP leads to photoreceptor dysfunction that phenocopies defects reported in patients with ARL2BP mutations .

Research shows that by P210 (210 days postnatal), ARL2BP knockout mice exhibit significant loss of photoreceptor nuclei, indicating degeneration of photoreceptor cells. This correlates with a 90% reduction in electroretinogram (ERG) response, confirming that ARL2BP is required for photoreceptor survival .

What are the key differences between various commercially available ARL2BP antibodies?

Different ARL2BP antibodies have unique properties that make them suitable for specific research applications:

The choice of antibody depends on your specific experimental requirements, including detection method, species of interest, and cellular localization studies .

What is the subcellular localization pattern of ARL2BP in photoreceptor cells?

ARL2BP displays a distinctive punctate staining pattern in photoreceptor cells. Immunolocalization studies using monoclonal antibodies have revealed that ARL2BP localizes to the inner segment (IS), basal body (BB), and connecting cilium (CC) of photoreceptors . This localization pattern is consistent with immunoelectron microscopy findings reported in previous studies .

Understanding this specific localization pattern is crucial for designing experiments investigating ciliary and axonemal functions in photoreceptors, as ARL2BP's presence in these structures corresponds to its role in microtubule organization and axoneme extension .

What are the recommended protocols for using FITC-conjugated ARL2BP antibody in flow cytometry?

When using FITC-conjugated ARL2BP antibody (such as NBP2-71743F) for flow cytometry, researchers should follow these methodological guidelines:

• Cell Preparation: Fix cells with 4% paraformaldehyde and permeabilize with 90% methanol to allow antibody access to intracellular targets .

• Antibody Dilution: Though specific dilutions may vary by manufacturer, a starting dilution of 1:200 is generally recommended. Always titrate the antibody to determine optimal concentration for your specific cell type .

• Controls: Include appropriate isotype controls (e.g., mouse IgG1 for NBP2-71743F) to establish baseline fluorescence and confirm specificity .

• Incubation: Incubate cells with the diluted antibody for 30-60 minutes at room temperature or overnight at 4°C in the dark to prevent photobleaching of the FITC fluorophore.

• Washing: Wash cells thoroughly with PBS containing 0.05-0.1% Tween-20 to remove unbound antibody before analysis.

For quantitative assessment, analyze samples on a flow cytometer with appropriate filters for FITC detection (typically 488nm excitation, 530/30nm emission) .

How can ARL2BP antibodies be used to investigate photoreceptor ciliary structure?

ARL2BP antibodies are valuable tools for investigating photoreceptor ciliary structure through several methodological approaches:

• Immunofluorescence Microscopy: Using FITC-conjugated ARL2BP antibodies allows direct visualization of ARL2BP localization in photoreceptor connecting cilium and basal body regions. This technique can reveal the punctate distribution pattern characteristic of ARL2BP .

• Co-localization Studies: ARL2BP antibodies can be used alongside markers for axonemal structures (such as RP1) to investigate protein interactions within the ciliary compartment. Research has shown that ARL2BP knockout affects the localization of other ciliary proteins like RPGR .

• Comparative Analysis: By comparing ARL2BP staining patterns between wild-type and disease model tissues, researchers can assess how mutations or treatments affect ciliary structure. For example, studies have demonstrated that ARL2BP knockout leads to defects in inner junction B-tubule closure in photoreceptor axonemes .

• Developmental Studies: Temporal analysis of ARL2BP localization during retinal development can provide insights into ciliary formation processes and the timing of potential therapeutic interventions.

These approaches have revealed that ARL2BP is critical for microtubule doublet formation and axoneme extension in photoreceptors, with implications for understanding ciliopathies and retinal degenerative disorders .

How can researchers validate the specificity of FITC-conjugated ARL2BP antibody staining?

Validating the specificity of FITC-conjugated ARL2BP antibody staining requires multiple complementary approaches:

• Knockout/Knockdown Controls: The most definitive validation method is testing the antibody in ARL2BP knockout or knockdown samples. Studies have confirmed absence of signal when using validated ARL2BP antibodies in knockout models, such as the ARL2BP knockout HeLa cell line (ab265269) .

• Peptide Competition Assays: Pre-incubating the antibody with excess immunizing peptide should block specific binding sites and eliminate true positive signal.

• Multiple Antibody Validation: Compare staining patterns from different ARL2BP antibodies raised against distinct epitopes. Concordant staining patterns increase confidence in specificity.

• Western Blot Correlation: Confirm that FITC-conjugated antibody detects a single band of the expected molecular weight (approximately 19-21 kDa) in western blots from the same samples used for immunofluorescence .

• Positive and Negative Tissue Controls: Include tissues known to express ARL2BP (e.g., A549 cells, photoreceptors) and those with minimal expression as controls in each experiment .

The expected band size for ARL2BP is approximately 19 kDa, though observed bands may appear at 20-21 kDa due to post-translational modifications .

What are common issues when using FITC-conjugated antibodies and how can they be addressed?

When working with FITC-conjugated ARL2BP antibodies, researchers may encounter several technical challenges:

• Photobleaching: FITC is susceptible to photobleaching, which can reduce signal intensity during imaging.
  Solution: Minimize exposure to light during all steps. Use anti-fade mounting media containing compounds like p-phenylenediamine or propyl gallate. Consider image acquisition strategies that minimize excitation light exposure.

• Autofluorescence: Cellular components, particularly in retinal tissue, can exhibit green autofluorescence that overlaps with FITC emission.
  Solution: Include unstained controls to assess background autofluorescence. Consider using Sudan Black B (0.1-0.3%) treatment to reduce autofluorescence or implement spectral unmixing during image analysis.

• pH Sensitivity: FITC fluorescence is optimal at alkaline pH (7.5-9.0) and decreases at lower pH values.
  Solution: Ensure buffers are maintained at optimal pH. For fixed samples, use PBS or TBS buffers at pH 7.4-8.0.

• Fixation-Related Issues: Overfixation can mask epitopes and reduce antibody binding.
  Solution: Optimize fixation protocols; for ARL2BP detection in cultured cells, 4% paraformaldehyde for 10-15 minutes is typically sufficient .

• Storage Stability: FITC conjugates may lose fluorescence intensity during storage.
  Solution: Store FITC-conjugated antibodies at 4°C in the dark, with appropriate preservatives as recommended by the manufacturer (typically PBS with 0.05% sodium azide) .

How can ARL2BP antibodies be used to investigate the relationship between ciliary dysfunction and retinitis pigmentosa?

ARL2BP antibodies serve as critical tools for investigating the mechanistic link between ciliary dysfunction and retinitis pigmentosa through several advanced methodological approaches:

• High-Resolution Ciliary Structural Analysis: Combining ARL2BP immunolabeling with super-resolution microscopy techniques (STED, STORM, or SIM) enables precise localization of ARL2BP within ciliary subdomains. This approach has revealed that ARL2BP is required for proper B-tubule closure in microtubule doublets of photoreceptor axonemes .

• Temporal Disease Progression Studies: Sequential immunohistochemical analysis using ARL2BP antibodies at different timepoints in animal models can track ciliary structural changes preceding photoreceptor degeneration. Research shows that by P210, ARL2BP knockout mice display significant photoreceptor nuclei loss, correlating with reduced ERG response .

• Multi-protein Interaction Analysis: Co-immunoprecipitation using ARL2BP antibodies followed by mass spectrometry can identify novel protein interactions within the ciliary compartment. This approach has helped establish that ARL2BP interacts with proteins involved in axoneme extension and disk morphogenesis .

• Comparative Analysis of Patient-Derived Samples: Immunofluorescence studies using ARL2BP antibodies on retinal organoids derived from patient iPSCs can validate disease mechanisms identified in animal models. This translational approach helps confirm that findings in knockout mice phenocopy defects observed in patients with ARL2BP mutations .

These sophisticated applications of ARL2BP antibodies have established that abnormal doublet structure in ARL2BP-deficient photoreceptors likely leads to impaired extension of the distal axoneme, compromising disk morphogenesis and outer segment structure—a central mechanism in certain forms of retinitis pigmentosa .

What techniques can be employed to study ARL2BP's role in microtubule organization and axoneme extension?

Investigating ARL2BP's specific role in microtubule organization and axoneme extension requires sophisticated methodological approaches:

• Correlative Light and Electron Microscopy (CLEM): This technique combines immunofluorescence using FITC-conjugated ARL2BP antibodies with transmission electron microscopy of the same specimen. This approach has revealed that ARL2BP knockout affects the transition from microtubule doublets to singlets in distal regions of photoreceptor axonemes .

• In vitro Microtubule Assembly Assays: Purified ARL2BP protein (detected and validated using specific antibodies) can be assessed for its direct effects on tubulin polymerization kinetics and microtubule stability in reconstitution experiments.

• Live Imaging of Axoneme Extension: Combining CRISPR-mediated tagging of endogenous ARL2BP with fluorescent reporters and live confocal imaging allows real-time visualization of axoneme extension processes in cellular models.

• Proximity Labeling Techniques: Methods such as BioID or APEX2 fused to ARL2BP can identify proximal proteins within the axonemal compartment, helping elucidate the molecular environment where ARL2BP functions.

• Cryo-Electron Tomography: This technique can be used following immunogold labeling with ARL2BP antibodies to visualize the three-dimensional architecture of axonemal microtubules and ARL2BP's precise positioning relative to doublet structures.

Studies employing these approaches have established that ARL2BP is specifically required for inner junction B-tubule closure in photoreceptor doublet microtubules, a critical step for proper axoneme extension and subsequent outer segment formation .

How does the performance of FITC-conjugated ARL2BP antibodies compare with other detection methods?

When evaluating FITC-conjugated ARL2BP antibodies against alternative detection methods, researchers should consider several performance parameters:

For quantitative studies requiring high sensitivity or extended imaging sessions, newer generation fluorophores like Alexa Fluor 488 may offer superior performance. For multicolor imaging experiments, careful selection of complementary fluorophores is essential to minimize spectral overlap with FITC's emission spectrum .

How can ARL2BP antibody staining be integrated with other cellular markers to investigate ciliary compartmentalization?

Integration of ARL2BP antibody staining with other cellular markers enables sophisticated analysis of ciliary compartmentalization through several methodological approaches:

• Multi-marker Immunofluorescence Protocols:

  • For FITC-conjugated ARL2BP antibodies, combine with spectrally distinct markers such as:

    • Acetylated α-tubulin (red channel) - labels entire axoneme

    • Pericentrin (far-red channel) - marks basal body

    • RP1 (red channel) - labels distal axoneme where singlet microtubules predominate

  • This approach has revealed that ARL2BP knockout affects the localization of RPGR and RP1 in photoreceptors .

• Sequential Immunodetection Strategy:

  • For densely packed structures like photoreceptor cilia, sequential staining with careful elution between rounds can overcome spatial detection limitations.

  • Protocol: Apply FITC-ARL2BP antibody first, image, elute (0.2M glycine, pH 2.5), verify elution, then apply next marker.

• Expansion Microscopy Integration:

  • Physical expansion of specimens using polymer embedding can spatially separate ciliary components for improved resolution.

  • FITC-conjugated antibodies are compatible with expansion protocols after appropriate fixation and permeabilization.

• Proximity Ligation Assay (PLA) Combinations:

  • Combining ARL2BP antibodies with antibodies against potential interaction partners in PLA reactions can visualize protein-protein interactions within ciliary compartments.

  • This technique generates fluorescent signals only when proteins are within 40nm proximity.

These integrated approaches have established that ARL2BP localizes to specific ciliary compartments and interacts with proteins essential for photoreceptor axoneme extension and outer segment formation, providing mechanistic insights into its role in retinal health and disease .