LRRC45 Antibody

What is LRRC45 and why is it important to study?

LRRC45 is a component of the proteinaceous fiber-like linker between two centrioles, required for centrosome cohesion. Its importance stems from its role in maintaining genomic stability across cell generations by stabilizing microtubules, ensuring proper anchoring of spindle fibers, and mediating centrosome cohesion . LRRC45 also plays a critical role in cilia biogenesis by organizing centriolar satellites and promoting the docking of Rab8 GTPase-positive vesicles, rather than merely serving as a recruitment platform for early vesicles . Research indicates LRRC45 associates with the distal appendages of the mother centriole in a Cep83 and SCLT1-dependent manner, contributing to cilia formation independently of C-Nap1 .

What are the commonly available types of LRRC45 antibodies?

Most commercially available LRRC45 antibodies are rabbit polyclonal antibodies. These antibodies typically:

The antibodies are raised against specific regions of the LRRC45 protein, with some targeting the C-terminal region (aa 615-643) and others targeting recombinant fragments within amino acids 350-550 or specific sequences like "RHSIINALKAKLQMTEAALALSEQKAQDLGELLATAEQEQLSLSQRQAKELKLEQQEAAERESKLLRDLSAANEKNLLLQNQVDELERKFRCQQE" .

What experimental applications are LRRC45 antibodies validated for?

LRRC45 antibodies have been validated for multiple experimental applications with specific recommended dilutions:

For optimal results, it's recommended to start with these dilutions and optimize according to your specific experimental conditions .

How should LRRC45 antibodies be stored and handled?

For short-term storage, maintain LRRC45 antibodies refrigerated at 2-8°C for up to 2 weeks. For long-term storage, store at -20°C in small aliquots to prevent freeze-thaw cycles which can degrade antibody performance . Most LRRC45 antibodies are supplied in buffered aqueous glycerol solution or PBS with 0.09% (W/V) sodium azide . When shipping these antibodies, use wet ice to maintain integrity .

How does the localization of LRRC45 change during the cell cycle and ciliogenesis?

During ciliogenesis, LRRC45 associates with the basal body of primary cilia in:

• Retina pigment epithelial (RPE1) cells

• Murine fibroblasts

• Neural stem cells

• Ependymal cells with motile cilia

This diverse localization pattern across cell types, including cells with motile cilia, suggests LRRC45 has a conserved function in promoting ciliogenesis beyond its role in centrosome cohesion .

What are the methodological considerations for investigating LRRC45 knockout/knockdown effects?

When designing LRRC45 depletion experiments, several methodological considerations are critical:

• Specificity of depletion: Use multiple independent siRNAs to confirm specificity of phenotypes. Research has verified specific effects using three independent siRNAs and a pool of 4 siRNAs targeting LRRC45 .

• Rescue experiments: Include rescue experiments to confirm specificity, as demonstrated in studies where ciliogenesis defects caused by LRRC45 depletion were rescued by reintroduction of LRRC45 .

• Phenotypic analysis: When investigating LRRC45 knockdown, examine:

  • Centrosome cohesion (primary function)

  • Cilia formation (independent function from centrosome cohesion)

  • Centriolar satellite organization

  • Vesicle docking during ciliogenesis

• Differentiate from other functions: Design experiments to distinguish LRRC45's role in ciliogenesis from its role in centrosome cohesion, such as comparing effects of LRRC45 depletion with depletion of C-Nap1 (a protein that recruits LRRC45 to the proximal end of centrioles) .

How can researchers resolve inconsistent immunostaining results with LRRC45 antibodies?

Inconsistent immunostaining results with LRRC45 antibodies can be resolved through several approaches:

• Antibody validation: Confirm antibody specificity using:

  • Protein arrays (e.g., arrays of 364 human recombinant protein fragments used to validate Prestige Antibodies)

  • Tissue arrays (e.g., 44 normal human tissues and 20 common cancer types)

  • LRRC45 knockout or knockdown controls

• Enhanced validation techniques: Look for antibodies validated through:

  • Orthogonal RNAseq validation

  • Independent antibody validation

  • Recombinant expression validation

• Fixation and permeabilization optimization: Test different protocols as LRRC45 localization to centrosomes and cilia may require specific conditions to preserve structure while allowing antibody access.

• Cross-validation with fluorescent protein fusions: Compare antibody staining patterns with LRRC45-GFP fusion proteins to verify localization patterns.

• Technical considerations table:

What experimental approaches can distinguish between LRRC45's centrosomal and ciliary functions?

To distinguish between LRRC45's centrosomal cohesion role and its ciliary functions, researchers can employ the following approaches:

• Domain-specific mutant expression: Generate truncated or point-mutated LRRC45 constructs targeting specific domains to identify which regions are required for centrosome cohesion versus cilia formation.

• Temporal analysis: Examine LRRC45 function at different cell cycle stages, as centrosome cohesion is critical during interphase while cilia formation occurs primarily in G0/G1.

• Co-depletion studies: Perform simultaneous knockdown of LRRC45 with either:

  • C-Nap1 (affects LRRC45 recruitment to proximal centrioles)

  • Cep83 or SCLT1 (affects LRRC45 association with distal appendages)

• Proximity labeling approaches: Use BioID or APEX2 proximity labeling with LRRC45 to identify distinct protein interaction networks associated with its centrosomal versus ciliary functions.

• Cell type-specific analysis: Compare LRRC45 functions in:

  • Cycling cells (where centrosome cohesion is critical)

  • Differentiated cells with stable cilia (where ciliary functions predominate)

  • Cells with motile cilia (ependymal cells) versus primary cilia (RPE1 cells)

What are the optimal parameters for using LRRC45 antibodies in Western blotting applications?

For optimal Western blot results with LRRC45 antibodies, consider the following parameters:

For improved results, consider:

• Using gradient gels (4-12%) for better resolution around the 76 kDa range

• Including protease inhibitors in lysis buffers to prevent degradation

• Running a pre-stained molecular weight marker adjacent to samples for accurate size determination

How can researchers optimize LRRC45 immunofluorescence staining for centrosome and cilia visualization?

Optimizing LRRC45 immunofluorescence for centrosome and cilia visualization requires attention to several technical details:

• Fixation protocol optimization:

  • For centrosome visualization: 4% paraformaldehyde (10 minutes) followed by methanol (-20°C, 5 minutes)

  • For cilia visualization: Methanol fixation alone often preserves ciliary structures better

• Co-staining markers:

  • For centrosomes: Use γ-tubulin or pericentrin as centrosome markers

  • For cilia: Use acetylated tubulin or Arl13b as axoneme markers

  • For basal bodies: Use Cep164 as a distal appendage marker

• Signal amplification techniques:

  • Consider tyramide signal amplification for weak signals

  • Use high-sensitivity detection systems for clearer visualization of discrete structures

• Imaging considerations:

  • Use confocal microscopy with z-stacking to fully capture 3D structures

  • Super-resolution techniques (SIM, STORM, STED) may provide better resolution of substructures

• Sample preparation recommendations:

What quality control measures should be implemented when validating a new batch of LRRC45 antibody?

Implementing rigorous quality control measures when validating new LRRC45 antibody batches ensures experimental reliability:

• Basic validation:

  • Western blot against positive control lysates (RT4, U251 MG cell lines)

  • Immunofluorescence on cells known to express LRRC45

  • Comparison with previous batches if available

• Specificity controls:

  • Peptide blocking experiments using the immunizing peptide

  • LRRC45 knockout/knockdown cells as negative controls

  • Cross-reactivity assessment with similar leucine-rich repeat proteins

• Functional validation:

  • Verify correct localization pattern (centrosome, cilia basal body)

  • Confirm expected molecular weight (76 kDa)

  • Test across multiple applications if the antibody is intended for multiple uses

• Documentation and standardization:

  • Record lot number, dilution optimization results, and imaging parameters

  • Establish standard positive controls for each application

  • Create a validation checklist specific to LRRC45 antibodies

• Comprehensive antibody validation table:

How can LRRC45 antibodies be used to investigate ciliopathy disease mechanisms?

LRRC45 antibodies can serve as valuable tools for investigating ciliopathy disease mechanisms through several research approaches:

• Patient sample analysis:

  • Compare LRRC45 expression and localization in patient-derived cells versus healthy controls

  • Examine correlation between LRRC45 abnormalities and ciliary defects in patient samples

• Disease modeling:

  • Use LRRC45 antibodies to assess centrosome and ciliary phenotypes in:

    • Patient-derived iPSCs and differentiated cells

    • Animal models of ciliopathies

    • CRISPR-engineered disease models

• Pathway analysis:

  • Investigate interaction between LRRC45 and known ciliopathy-associated proteins

  • Examine changes in LRRC45 localization upon disruption of ciliopathy gene function

• Tissue-specific analyses:

  • Use immunohistochemistry to examine LRRC45 in affected tissues from ciliopathy models

  • Compare LRRC45 patterns across multiple ciliated tissues (retina, kidney, brain)

• Research application matrix:

Given LRRC45's established presence at the basal body of both primary and motile cilia in various cell types, including retinal, neural, and ependymal cells, it represents a promising target for understanding ciliopathy mechanisms .

What approaches can reveal the temporal dynamics of LRRC45 during centrosome duplication and ciliogenesis?

To investigate the temporal dynamics of LRRC45 during centrosome duplication and ciliogenesis, researchers can employ:

• Live-cell imaging techniques:

  • LRRC45-GFP fusion protein expression for real-time tracking

  • Photoactivatable or photoswitchable LRRC45 fusions to track protein movement

  • Correlative light-electron microscopy to link dynamic behavior with ultrastructure

• Cell synchronization approaches:

  • Examine LRRC45 localization at specific cell cycle stages using:

    • Thymidine block (S-phase)

    • Nocodazole treatment (M-phase)

    • Serum starvation (G0/G1, ciliogenesis induction)

• Pulse-chase experiments:

  • Use SNAP-tag LRRC45 fusions with timed labeling to track protein turnover

  • Photobleaching recovery (FRAP) to measure protein dynamics at centrosomes/cilia

• Staged ciliogenesis analysis:

  • Fix cells at defined time points after serum starvation

  • Co-stain for LRRC45 alongside markers of sequential ciliogenesis stages:

    • CP110 removal

    • Rab8 vesicle docking

    • Axoneme extension

    • IFT establishment

• Temporal dynamics analysis table:

How can LRRC45 antibodies be combined with other markers to study centrosome-cilium relationship in disease states?

Combining LRRC45 antibodies with other markers provides powerful insights into centrosome-cilium abnormalities in disease:

• Multiplex immunofluorescence panels:

  • LRRC45 + centrosomal markers (γ-tubulin, pericentrin) + ciliary markers (acetylated tubulin, Arl13b)

  • Include markers for specific centrosomal structures: PCM (pericentrin), distal appendages (Cep164), subdistal appendages (ODF2)

  • Add cell cycle markers (Ki67, PCNA) to correlate with proliferation status

• Pathway-specific co-staining:

  • LRRC45 + Hedgehog pathway components (Gli, Smoothened)

  • LRRC45 + Wnt signaling components (Dishevelled, β-catenin)

  • LRRC45 + autophagy markers (LC3, p62) in ciliopathies with autophagy defects

• Structured illumination microscopy (SIM) application:

  • Resolve subdomains of basal body and transition zone in relation to LRRC45

  • Map precise LRRC45 localization relative to known disease-associated proteins

• Tissue microarray analysis:

  • Apply optimized LRRC45 antibody panels to disease tissue arrays

  • Compare patterns across multiple patient samples and disease types

• Marker combination matrix for disease analysis:

This multiplexed approach is particularly valuable given LRRC45's dual roles in centrosome cohesion and cilia formation, allowing researchers to determine which function is compromised in specific disease states .

How should researchers interpret discrepancies between LRRC45 antibody staining patterns in different cell types?

When facing discrepancies in LRRC45 staining patterns across cell types, consider these interpretive approaches:

• Biological versus technical variation assessment:

  • Verify antibody specificity in each cell type using knockdown controls

  • Test multiple LRRC45 antibodies targeting different epitopes

  • Consider cell type-specific post-translational modifications affecting epitope recognition

• Cell type-specific biology considerations:

  • Different cell types may express varying LRRC45 isoforms or interacting partners

  • Ciliation status varies dramatically between cell types (e.g., RPE1 vs. neural stem cells vs. ependymal cells)

  • Cell cycle distribution differences affect centrosome cohesion requirements

• Quantitative approach to pattern comparison:

  • Measure signal intensity at specific subcellular locations

  • Calculate the ratio of LRRC45 at different locations (proximal centrioles vs. distal appendages)

  • Track pattern changes during differentiation or disease progression

• Interpretation framework:

• Integrated analysis approach: Combine multiple techniques (IF, WB, IP-MS) to build a comprehensive picture of LRRC45 biology across cell types rather than relying on a single method .

What controls are essential when using LRRC45 antibodies to investigate centrosome abnormalities in cancer?

When investigating centrosome abnormalities in cancer using LRRC45 antibodies, implement these essential controls:

• Antibody validation controls:

  • Positive control: Known LRRC45-expressing cells (e.g., RT4, U251 MG)

  • Negative control: LRRC45 knockdown/knockout cells

  • Signal specificity: Secondary-only and isotype controls

• Sample preparation controls:

  • Matched normal tissue adjacent to tumor

  • Non-malignant cell line of same tissue origin

  • Panel of cancer cell lines with characterized centrosome abnormalities

• Biological interpretation controls:

  • Cell cycle markers to distinguish cycle-dependent variations

  • Centrosome amplification markers (e.g., extra centrin dots)

  • DNA damage markers to correlate with genomic instability

• Cancer-specific control panel:

• Experimental design considerations:

  • Include tissue microarrays with multiple cancer types

  • Compare primary tumors with metastatic samples

  • Assess therapy effects on LRRC45 patterns in paired pre/post-treatment samples

The critical importance of proper controls is underscored by LRRC45's role in centrosome cohesion, as centrosome abnormalities are hallmarks of many cancers and contribute to genomic instability .

How can researchers distinguish specific LRRC45 signals from common artifacts in immunostaining experiments?

Distinguishing genuine LRRC45 signals from artifacts requires systematic troubleshooting:

• Common artifact patterns and solutions:

• Signal validation approaches:

  • Confirm expected subcellular localization with co-staining:

    • LRRC45 should colocalize with centrosomal markers at centriole linker

    • LRRC45 should associate with distal appendages in a Cep83/SCLT1-dependent manner

  • Verify signal disappearance following LRRC45 depletion

  • Check for expected cell cycle-dependent localization changes

• Quantitative validation:

  • Measure signal-to-noise ratio in controlled samples

  • Compare staining intensity across multiple antibody lots

  • Establish threshold values for positive versus background signal

• Advanced troubleshooting techniques:

  • Epitope retrieval optimization for each tissue/cell type

  • Signal amplification methods for weak but specific signals

  • Super-resolution microscopy to resolve genuine centrosomal structures

• Decision tree for artifact determination:

  • Does signal localize to expected structures (centrosomes, basal bodies)?

  • Does signal disappear in knockdown/knockout controls?

  • Is the pattern consistent with published LRRC45 localization data?

  • Does the pattern change appropriately during cell cycle progression?

  • Is the molecular weight correct in parallel Western blot analysis?

Using these systematic approaches, researchers can confidently distinguish genuine LRRC45 signals from common immunostaining artifacts.