rilpl1 Antibody

What are the validated applications for RILPL1 antibody in experimental settings?

RILPL1 antibodies have been validated for several laboratory applications, with Western Blot (WB), Immunofluorescence (IF), and ELISA being the most commonly utilized. According to product validation data, RILPL1 antibodies such as 16732-1-AP have demonstrated reliable detection in WB, IF, and ELISA applications . For researchers planning experimental design, it's important to note that different antibody clones may have varying application strengths - for example, antibody 83220-6-RR has been primarily validated for WB and ELISA applications . When designing experiments, researchers should select antibodies specifically validated for their intended application to ensure reliable results.

What is the optimal dilution range for RILPL1 antibody in Western blot applications?

The recommended dilution range for RILPL1 antibody varies by clone and application. For Western blot applications, polyclonal antibody 16732-1-AP works effectively at dilutions between 1:500-1:1000 . In contrast, the recombinant RILPL1 antibody 83220-6-RR demonstrates higher sensitivity and can be used at significantly higher dilutions, ranging from 1:5000 to 1:50000 for Western blot applications . Researchers should note that optimal dilution may be sample-dependent, requiring titration in specific experimental systems to determine ideal conditions for maximum sensitivity while minimizing background signal.

What species reactivity has been confirmed for commercially available RILPL1 antibodies?

Commercial RILPL1 antibodies have been tested and validated for reactivity across multiple mammalian species. Both the 16732-1-AP and 83220-6-RR antibodies demonstrate confirmed reactivity with human, mouse, and rat samples . This cross-species reactivity enables comparative studies across different model organisms. In published research applications, human samples have been the most frequently cited for RILPL1 antibody applications . Researchers working with other species should perform validation experiments before proceeding with full-scale studies.

What is the expected molecular weight for RILPL1 detection in Western blot applications?

When performing Western blot analysis, researchers should note the discrepancy between the calculated and observed molecular weights for RILPL1. While the calculated molecular weight based on amino acid composition is 29 kDa (252 amino acids), the observed molecular weight in experimental conditions is consistently around 50 kDa . This difference may be attributed to post-translational modifications or structural characteristics affecting protein migration. For proper band identification, researchers should use positive controls such as lysates from HepG2 cells, HeLa cells, HEK-293 cells, or A549 cells, which have been validated to express detectable levels of RILPL1 .

How can researchers optimize immunofluorescence detection of RILPL1 in primary cilium structures?

Optimizing immunofluorescence detection of RILPL1 in primary cilium structures requires careful consideration of fixation methods and co-staining markers. Research has demonstrated that RILPL1 localizes to the primary cilium and centrosome, with specific localization to the distal end of the mother centriole . For successful visualization:

• Use paraformaldehyde fixation (4%) to preserve ciliary structures

• Include co-staining with ciliary markers such as acetylated tubulin (acTub) and centrosomal markers like γ-tubulin and pericentrin

• For higher resolution imaging of RILPL1's specific localization at the mother centriole, include distal appendage markers such as Cep164

• Consider deconvolution microscopy for precise suborganelle localization analysis

Researchers should note that RILPL1 localization is cell cycle dependent, with approximately 70% of cells showing centriolar localization during G1 phase, making cell synchronization potentially beneficial for consistent results .

What experimental approaches can differentiate between RILPL1 and RILPL2 when studying ciliary function?

Differentiating between RILPL1 and RILPL2 in ciliary function studies requires targeted experimental strategies due to their structural similarities and partially overlapping functions:

• Antibody selection: Use validated antibodies specific to unique sequences - polyclonal antibodies directed against sequences unique to RILPL1 have been validated to distinguish between the paralogs in immunofluorescence applications

• Localization analysis: While both proteins localize to primary cilia and centrosomes, RILPL1 specifically localizes to the distal end of the mother centriole, while RILPL2 shows less specific centrosomal subdomain localization

• Expression pattern analysis: RILPL2 is upregulated in multiciliated cells, while RILPL1 expression patterns differ, being found at one of two γ-tubulin foci in neighboring non-multiciliated cells

• Cell cycle analysis: RILPL1 shows distinct cell-cycle dependent localization patterns, remaining at only one centriole during G1 through prophase, disappearing from centrosomes during metaphase through anaphase, and reappearing at telophase/cytokinesis

These approaches, used individually or in combination, can effectively distinguish between these related proteins in experimental systems.

How should researchers approach RILPL1 antibody validation in LRRK2-dependent lysosomal recruitment studies?

When studying LRRK2-dependent recruitment of RILPL1 to lysosomes, comprehensive antibody validation is critical for reliable results. Based on recent research findings, a methodological approach should include:

• Orthogonal validation: Combine immunostaining with biochemical approaches to confirm RILPL1 recruitment to lysosomes. Endogenous RILPL1 has been successfully detected on LRRK2-positive lysosomes after lysosomal damage (e.g., LLOME treatment) in multiple cell types including HEK293T cells, U2OS cells, and mouse primary astrocytes

• Control conditions: Include LRRK2 kinase inhibition as a negative control condition, as RILPL1 recruitment to lysosomes is dependent on LRRK2 kinase activity

• Antibody specificity verification: Validate anti-RILPL1 antibodies specifically against RILPL1-knockout or knockdown samples to confirm specificity and rule out cross-reactivity

• Co-localization analyses: Use confocal microscopy with established lysosomal markers alongside LRRK2 and RILPL1 staining to quantify recruitment under various experimental conditions

This methodological framework ensures reliable detection of RILPL1 in the context of LRRK2-dependent lysosomal recruitment studies.

What storage and handling conditions are recommended to maintain RILPL1 antibody stability and performance?

Proper storage and handling of RILPL1 antibodies are essential for maintaining their stability and performance over time. Based on manufacturer recommendations, researchers should adhere to the following guidelines:

• Storage temperature: Store RILPL1 antibodies at -20°C, where they remain stable for one year after shipment

• Buffer composition: RILPL1 antibodies are typically supplied in PBS with 0.02% sodium azide and 50% glycerol at pH 7.3, which helps maintain stability

• Aliquoting considerations: For the standard formulation, aliquoting is unnecessary for -20°C storage, which simplifies laboratory handling

• Special formulations: Note that some smaller volume preparations (20μl) may contain 0.1% BSA as a stabilizing agent

• Freeze-thaw cycles: While the glycerol formulation provides some protection against freeze-thaw damage, minimizing repeated freeze-thaw cycles is still recommended for optimal antibody performance

Adherence to these storage and handling recommendations will ensure consistent antibody performance across experiments and over time.

How can researchers troubleshoot high background issues when using RILPL1 antibody in immunofluorescence applications?

High background signal in RILPL1 immunofluorescence can compromise data interpretation. To address this common issue, researchers should consider the following optimization approaches:

• Blocking optimization: Use 5-10% normal serum from the same species as the secondary antibody combined with 0.1-0.3% Triton X-100 for permeabilization and blocking

• Antibody dilution: For polyclonal RILPL1 antibodies, test more dilute solutions than recommended for Western blot applications, as immunofluorescence often requires higher dilutions to reduce background

• Secondary antibody selection: Use highly cross-adsorbed secondary antibodies to minimize non-specific binding, and include secondary-only controls to assess background contribution

• Washing protocol optimization: Implement extended washing steps (4-5 washes of 5-10 minutes each) with PBS containing 0.1% Tween-20 after both primary and secondary antibody incubations

• Sample fixation considerations: Test both paraformaldehyde (4%) and methanol fixation methods, as certain epitopes may be better preserved with specific fixation protocols

• Autofluorescence reduction: Include an autofluorescence quenching step, particularly important when working with tissues or cells with high intrinsic fluorescence

Implementation of these methodological refinements can significantly improve signal-to-noise ratio in RILPL1 immunofluorescence applications.

What are the recommended positive control samples for validating RILPL1 antibody performance?

When validating RILPL1 antibody performance, selecting appropriate positive control samples is crucial for establishing experimental reliability. Based on validated detection data, the following samples serve as effective positive controls:

For immunofluorescence applications specifically studying cilia, mouse tracheal epithelial cells (MTECs) have been validated for endogenous RILPL1 detection and serve as excellent controls for ciliary/centrosomal localization . When analyzing subcellular localization, NIH 3T3 or N2A cells can serve as reliable models for centrosomal and mother centriole localization studies .

How does RILPL1 localization change throughout the cell cycle, and what methodological approaches best capture these dynamics?

RILPL1 exhibits distinct localization patterns throughout the cell cycle, requiring specific methodological approaches to accurately document these dynamics:

• G1 phase: Approximately 70% of cells show RILPL1 localization to a single centriole (the mother centriole)

• S/G2 phase: Following centriole duplication, RILPL1 remains localized to only one of four centrioles

• Prophase: RILPL1 maintains localization to only one centriole

• Metaphase through anaphase: RILPL1 disappears from both centrosomes at the spindle poles

• Telophase/cytokinesis: RILPL1 reappears as one focus at the centrosomes of each new daughter cell

To effectively capture these dynamics, researchers should employ:

• Cell synchronization protocols to enrich for specific cell cycle stages

• Co-staining with cell cycle markers (e.g., cyclin proteins) to definitively identify cell cycle phases

• Live-cell imaging with fluorescently tagged RILPL1 (e.g., LAP-tagged RILPL1) for continuous monitoring

• Deconvolution microscopy for high-resolution localization analysis

• Co-staining with mother centriole markers (e.g., Cep164) to confirm specific localization patterns

This combined methodological approach provides comprehensive documentation of RILPL1's cell cycle-dependent localization dynamics.

What is the role of RILPL1 in ciliary membrane protein regulation, and how can researchers experimentally assess this function?

RILPL1 plays a critical role in regulating protein localization in the primary cilium, specifically in controlling ciliary membrane protein concentration by promoting protein removal from the primary cilium . To experimentally assess this function, researchers can employ several methodological approaches:

• Depletion studies: siRNA or CRISPR-based depletion of RILPL1 results in accumulation of signaling proteins in the ciliary membrane, providing a direct readout of its regulatory function

• Three-dimensional cell culture: Loss of RILPL1 prevents proper epithelial cell organization in 3D culture systems, offering a functional assessment of its role in cellular architecture maintenance

• Live-cell microscopy: This approach reveals that RILPL1 family proteins show dynamic localization to the primary cilium and association with tubulovesicular structures at the cilium base, providing insight into trafficking mechanisms

• Protein domain analysis: Functional studies separating the RH1 domain (aa 1-288) from the RH2 domain (aa 289-406) can help dissect which protein regions are responsible for specific aspects of ciliary regulation

• Co-immunoprecipitation studies: These can identify interaction partners involved in the ciliary protein removal pathway

These experimental approaches provide complementary insights into RILPL1's role in ciliary membrane protein regulation and dynamics.

How does RILPL1 interact with LRRK2 and RAB proteins in lysosomal contexts, and what techniques best demonstrate these interactions?

Recent research has revealed important interactions between RILPL1, LRRK2, and RAB proteins in lysosomal contexts. RILPL1 is recruited to ruptured lysosomes via LRRK2 activity to promote phosphorylation of RAB proteins . To effectively demonstrate and study these interactions, researchers should consider the following methodological approaches:

• Lysosomal damage models: Utilize LLOME treatment to induce lysosomal damage, triggering RILPL1 recruitment in cellular models including HEK293T cells, U2OS cells, and primary mouse astrocytes

• LRRK2 inhibition studies: Employ specific LRRK2 kinase inhibitors as negative controls, as RILPL1 recruitment to lysosomes depends on LRRK2 kinase activity

• Immunofluorescence co-localization: Perform triple staining for RILPL1, LRRK2, and lysosomal markers to visualize recruitment dynamics

• Phospho-specific antibodies: Use antibodies detecting phosphorylated RAB proteins to correlate RILPL1 recruitment with downstream signaling events

• Proximity labeling techniques: BioID or APEX2-based proximity labeling can identify proteins in close proximity to RILPL1 in lysosomal contexts

• Super-resolution microscopy: Techniques such as STORM or STED microscopy can resolve the nanoscale organization of RILPL1, LRRK2, and RAB proteins at the lysosomal membrane

These complementary approaches provide a comprehensive toolkit for investigating the functional relationships between RILPL1, LRRK2, and RAB proteins in lysosomal contexts.

What is known about RILPL1's association with oculopharyngodistal myopathy, and how can researchers investigate this connection?

Publications indicate that CGG repeat expansion in RILPL1 is associated with oculopharyngodistal myopathy type 4 . Although detailed information is limited in the provided search results, researchers interested in investigating this disease association could employ the following methodological approaches:

• Genetic screening: Develop PCR-based or sequencing assays to detect CGG repeat expansions in the RILPL1 gene in patient cohorts

• Patient-derived cell models: Establish fibroblast or induced pluripotent stem cell (iPSC) lines from affected individuals to study cellular phenotypes

• Immunohistochemistry: Apply RILPL1 antibodies to muscle biopsy specimens from patients to assess protein localization and abundance in disease contexts

• Functional studies: Create cellular or animal models with the disease-associated repeat expansion to evaluate effects on RILPL1 expression, localization, and function

• Proteomics approaches: Identify altered protein interactions or post-translational modifications of RILPL1 in the context of the disease-associated mutation

This multi-faceted approach would enable researchers to investigate the molecular mechanisms underlying RILPL1's role in oculopharyngodistal myopathy.

What methodological considerations are important when studying the differences between RILPL1 and RILPL2 in experimental systems?

While RILPL1 and RILPL2 share structural similarities and both regulate protein localization in primary cilia, they exhibit distinct expression patterns and subcellular localizations that require careful methodological considerations for differentiation:

• Expression analysis: RILPL2 is specifically upregulated in multiciliated cells, while RILPL1 shows different expression patterns across cell types, providing an opportunity for differential analysis in mixed cell populations

• Domain-specific tools: Design experiments targeting the specific domains of each protein - both contain RH1 and RH2 domains, but with distinct functional properties that can be exploited for differential analysis

• Localization precision: While both proteins localize to cilia and centrosomes, RILPL1 specifically localizes to the distal end of the mother centriole, requiring high-resolution imaging techniques like deconvolution microscopy with appropriate markers (e.g., Cep164) for proper distinction

• Temporal dynamics: RILPL1 shows specific cell cycle-dependent localization patterns, providing a temporal dimension for differentiation

• Specific antibody validation: Use antibodies directed against unique sequences in each protein, with careful validation against overexpression and knockdown controls to ensure specificity

These methodological considerations help researchers accurately distinguish between these related proteins in experimental systems, enabling precise functional characterization of each.

What emerging technologies might enhance RILPL1 detection and functional analysis in future research?

Emerging technologies offer promising avenues for advancing RILPL1 detection and functional analysis:

• Super-resolution microscopy: Techniques such as STORM, PALM, or lattice light-sheet microscopy could provide unprecedented spatial resolution of RILPL1's precise localization at the distal end of the mother centriole and within primary cilia

• Live-cell quantitative phase imaging: This allows for label-free, long-term observation of cellular dynamics related to RILPL1 function without phototoxicity concerns

• Proximity labeling advancements: Next-generation proximity labeling approaches like TurboID or Split-TurboID could map RILPL1's dynamic protein interaction network with improved temporal resolution

• CRISPR-based endogenous tagging: CRISPR knock-in of fluorescent or affinity tags at the endogenous RILPL1 locus would enable physiological level visualization and purification

• Single-molecule tracking: This would reveal the kinetics and dynamics of individual RILPL1 molecules within living cells, providing insights into its movement between subcellular compartments

• Spatial transcriptomics and proteomics: These approaches could map RILPL1's expression and interaction patterns across tissues and subcellular compartments with unprecedented resolution