CELSR1 Antibody, FITC conjugated

What is CELSR1 and what cellular functions does it mediate?

CELSR1 (Cadherin EGF LAG seven-pass G-type receptor 1) is a member of the flamingo subfamily within the cadherin superfamily. Unlike classic cadherins, CELSR1 does not interact with catenins and possesses a unique structure comprising nine cadherin domains, seven epidermal growth factor-like repeats, and two laminin A G-type repeats in the ectodomain. The protein also contains seven transmembrane domains, a signature characteristic of this subfamily . CELSR1 functions as a receptor in contact-mediated cell communication, with its cadherin domains serving as homophilic binding regions and the EGF-like domains participating in cell adhesion and receptor-ligand interactions . It plays a critical role in planar cell polarity (PCP) and is essential for organizing asymmetric protein complexes at cell junctions . As a developmentally regulated, neural-specific gene, CELSR1 has significant involvement in early embryogenesis .

What are the recommended storage conditions for CELSR1 antibody, FITC conjugated?

The CELSR1 polyclonal antibody with FITC conjugation should be stored at -20°C in its provided aqueous buffered solution (0.01M TBS, pH 7.4, with 1% BSA, 0.03% Proclin300, and 50% Glycerol) . To maintain antibody integrity and performance, it is strongly recommended to aliquot the antibody into multiple small volumes before freezing, thereby avoiding repeated freeze-thaw cycles that can degrade antibody quality . When properly stored, the antibody maintains its immunoreactivity for the intended applications without significant loss of signal intensity.

What species reactivity does the CELSR1 polyclonal antibody demonstrate?

The CELSR1 polyclonal antibody exhibits reactivity across multiple mammalian species, specifically human, mouse, and rat samples . This cross-species reactivity derives from the immunogen design, which utilized a KLH-conjugated synthetic peptide derived from human CELSR1 corresponding to amino acid residues 751-850 within the full 3014 amino acid sequence . The preserved epitope regions across these species allow researchers to use the same antibody preparation for comparative studies between human samples and common rodent models, facilitating translational research approaches.

What is the optimal dilution range for immunofluorescence applications with CELSR1-FITC antibody?

For immunofluorescence (IF) or immunocytochemistry (ICC) applications, the recommended dilution range for the CELSR1 polyclonal antibody (FITC conjugated) is 1:50-1:200 . The optimal dilution should be determined empirically for each specific application and cell type, as factors such as target protein expression levels, fixation methods, and detection sensitivity can influence the signal-to-noise ratio. Begin with a mid-range dilution (1:100) and adjust based on signal intensity and background levels. Importantly, when comparing expression levels between different experimental conditions, maintain a consistent antibody dilution across all samples to ensure valid quantitative comparisons.

How can CELSR1 antibody be used to investigate trans- versus cis-interactions in cell aggregation assays?

To investigate CELSR1's trans- versus cis-interactions using CELSR1-FITC antibody, researchers can employ cell aggregation assays with specific experimental controls. For trans-interaction studies, express CELSR1 in two separate HEK293T cell populations, co-express with different fluorescent reporters (e.g., GFP and mCherry), then mix the cells and quantify aggregation patterns . The CELSR1-FITC antibody can be used to validate expression levels prior to mixing. For assessing calcium dependence of trans-interactions, include EGTA (calcium chelator) controls, which should abolish CELSR1-mediated aggregation but not affect control proteins like TEN2/ADGRL3 .

To distinguish cis-interactions, compare wild-type CELSR1 with modified constructs (e.g., ΔCADH1-8 or ΔCADH9-GAIN) using both cell aggregation and junction enrichment assays . In junction enrichment assays, co-stain for CELSR1 (using the FITC-conjugated antibody) and cell junction markers like ZO-1 to assess localization patterns. Stable junctional enrichment with punctate distribution suggests functional cis-interactions, while diffuse surface distribution indicates impaired lateral interactions . FRAP (Fluorescence Recovery After Photobleaching) analysis can further quantify mobility differences between wild-type and mutant proteins, with increased mobility suggesting compromised cis-interactions .

What experimental approaches can be used to study CELSR1's role in planar cell polarity (PCP) organization?

To investigate CELSR1's role in planar cell polarity organization, researchers can employ multiple complementary approaches:

• Super-resolution microscopy: Use techniques like Structured Illumination Microscopy (SIM) to visualize asymmetric distribution of CELSR1 and its binding partners (Frizzled6 and Vangl2) across cell junctions . The FITC-conjugated CELSR1 antibody can be paired with differently labeled antibodies against PCP proteins to analyze their spatial relationships.

• Mutant analysis: Compare wild-type CELSR1 with known PCP-disrupting mutants like Celsr1^Crsh^ to assess how specific domains contribute to protein function . Examine the mutant's ability to form stable junctions, enrich at cell borders, and organize PCP proteins.

• Rescue experiments: Perform rescue experiments using ectopic cis-dimerization strategies to restore function to mutant CELSR1 variants, confirming the specific role of lateral interactions in PCP organization .

• Biochemical interaction studies: Combine these approaches with co-immunoprecipitation assays to determine if CELSR1 physically interacts with other PCP proteins and how mutations affect these interactions .

• Live cell imaging: Track the dynamics of CELSR1-mediated adhesion complex formation using FRAP and time-lapse microscopy to understand the temporal aspects of PCP establishment .

This multi-faceted approach can reveal how CELSR1's adhesive properties contribute to the molecular asymmetry required for proper tissue polarization.

How can researchers distinguish between specific and non-specific staining when using CELSR1-FITC antibody?

To distinguish between specific and non-specific staining when using CELSR1-FITC antibody, researchers should implement several validation controls:

• Negative controls: Include samples where the primary antibody is omitted but all other steps remain identical. This reveals background fluorescence from non-specific binding of secondary reagents (if used) or autofluorescence.

• Blocking peptide competition: Pre-incubate the CELSR1-FITC antibody with excess immunizing peptide (residues 751-850 of human CELSR1) before applying to samples. Specific staining should be significantly reduced or eliminated.

• Expression controls: Compare staining patterns between cells/tissues known to express high versus low levels of CELSR1, or use CELSR1 knockout/knockdown models as negative controls.

• Subcellular localization assessment: Verify that staining patterns match the expected plasma membrane localization with enrichment at cell-cell junctions for wild-type CELSR1 .

• Co-localization studies: Confirm partial co-localization with known CELSR1 interaction partners like Frizzled6 and Vangl2 at cell junctions .

True specific staining should be consistent with CELSR1's biological properties: enrichment at cell-cell contacts, punctate distribution patterns, and asymmetric localization in polarized tissues.

How should researchers interpret differences in CELSR1 staining patterns between wild-type and mutant samples?

When interpreting differences in CELSR1 staining patterns between wild-type and mutant samples, researchers should consider several key parameters:

• Subcellular distribution: Wild-type CELSR1 typically exhibits enrichment at cell borders with organized punctate assemblies, while mutants like Celsr1^Crsh^ often show diffuse cell surface distribution with reduced junctional enrichment . This pattern difference suggests impaired adhesive interactions rather than complete loss of function.

• Stability and dynamics: Assess FRAP recovery kinetics—wild-type CELSR1 shows stable junctional localization with limited mobility, while impaired cis-interaction mutants demonstrate increased mobility despite maintaining some trans-adhesion capability .

• PCP protein organization: Examine how CELSR1 mutations affect the asymmetric organization of PCP proteins like Frizzled6 and Vangl2. Although mutants may physically interact with these proteins, they often fail to organize them into proper asymmetric complexes .

• Interaction patterns: In mixed experiments with wild-type and mutant proteins, analyze whether heterotypic interactions occur. Some mutations selectively impair interactions with wild-type protein despite maintaining homotypic interactions .

• Calcium dependence: Verify whether adhesion remains calcium-dependent (abolished by EGTA), which is characteristic of cadherin-mediated interactions .

These interpretative frameworks help distinguish between mutations affecting protein expression/trafficking versus those specifically disrupting adhesive or signaling functions.

What factors might contribute to inconsistent results when using CELSR1-FITC antibody in immunofluorescence studies?

Several factors can contribute to inconsistent results when using CELSR1-FITC antibody in immunofluorescence studies:

• Fixation sensitivity: CELSR1, with its multiple transmembrane domains and complex tertiary structure, may be sensitive to fixation methods. Overfixation can mask epitopes while insufficient fixation may not preserve structure adequately. Test multiple fixation protocols (4% PFA, methanol, or gentle fixation with 2% PFA) to determine optimal conditions.

• Expression levels: Variable endogenous CELSR1 expression across cell types or developmental stages can affect detection sensitivity. Consider using signal amplification methods for low-expressing samples.

• Antibody degradation: FITC conjugates are susceptible to photobleaching and degradation over time. Minimize light exposure during storage and handling, and verify antibody performance regularly using positive controls.

• Calcium-dependent epitope conformation: Since CELSR1 adhesion is calcium-dependent , epitope accessibility may vary with calcium levels. Standardize sample preparation buffers to maintain consistent calcium concentration.

• Protein trafficking dynamics: CELSR1 localization changes during junctional maturation. Standardize the timing of analysis post-confluence or post-calcium switch in cell culture experiments.

• Interference from interaction partners: CELSR1's association with other proteins may mask epitopes in certain cellular contexts. Consider using multiple antibodies targeting different epitopes to validate findings.

To enhance reproducibility, maintain consistent protocols for cell culture conditions, fixation timing, antibody concentration, and imaging parameters across experiments.

How can CELSR1-FITC antibody be used to investigate the molecular mechanisms of planar cell polarity defects in developmental disorders?

CELSR1-FITC antibody can be instrumental in investigating planar cell polarity defects in developmental disorders through several advanced research approaches:

• Patient-derived tissue analysis: Apply the antibody to tissue samples from patients with neural tube defects, hair patterning abnormalities, or ciliopathies to evaluate CELSR1 distribution patterns compared to control tissues. Asymmetric localization defects may provide mechanistic insights into disease pathology.

• CRISPR-engineered disease models: Generate cell lines or animal models carrying specific CELSR1 mutations identified in human disorders using CRISPR/Cas9 technology. The FITC-conjugated antibody can then be used to visualize how these mutations affect protein localization, stability, and interaction with other PCP components like Frizzled6 and Vangl2 .

• Quantitative asymmetry analysis: Develop computational image analysis pipelines to quantify the degree of asymmetric CELSR1 distribution using structured illumination microscopy or other super-resolution techniques . This allows objective comparison between normal and pathological samples.

• Dynamic junctional complex assembly: Use live-cell imaging with complementary fluorescent tags to track how CELSR1 and its partners assemble during development, and identify precise temporal disruptions in disease models.

• Molecular rescue strategies: Test whether engineered cis-dimerization or other molecular interventions can rescue defective CELSR1 function in disease models, providing proof-of-concept for potential therapeutic approaches .

By systematically analyzing how disease-associated mutations affect CELSR1's adhesive properties and organizational capabilities, researchers can establish mechanistic links between molecular defects and developmental phenotypes.

What experimental design would best assess the roles of different CELSR1 domains in adhesive versus signaling functions?

An optimal experimental design to dissect CELSR1's adhesive versus signaling functions would employ a comprehensive domain-deletion and mutation strategy complemented by functional assays:

Based on existing research, this design would build upon findings that CADH1-8 is essential for cell-cell adhesion while mutations in the CADH9-GAIN interface enhance cellular adhesion without affecting signaling . Cell aggregation assays would quantify adhesive function, while junction enrichment assays with ZO-1 co-staining would assess junctional maturation . FRAP analysis would measure protein mobility as an indicator of stable complex formation .

For signaling function assessment, downstream PCP pathway activation could be monitored using established reporter systems. Importantly, all constructs should be normalized for cell-surface expression levels using surface biotinylation or flow cytometry to ensure accurate comparison . This comprehensive approach would establish whether specific domains contribute exclusively to adhesion or signaling, or whether they serve dual functions in CELSR1's complex biology.

How might heterophilic interactions between CELSR1 and CELSR2 affect experimental interpretations when using CELSR1-FITC antibody?

Heterophilic interactions between CELSR1 and CELSR2 create important considerations for experimental design and data interpretation when using CELSR1-FITC antibody:

• Antibody specificity verification: Cross-reactivity between CELSR family members must be rigorously tested. The FITC-conjugated CELSR1 antibody should be validated against cells expressing only CELSR1, only CELSR2, or both proteins to confirm selective labeling of CELSR1.

• Complex interpretation in native tissues: Since CELSR1 and CELSR2 can form heterophilic aggregates in calcium-dependent manner , staining patterns in tissues expressing both proteins may reflect heterotypic complexes rather than homotypic CELSR1 assemblies. This complicates interpretation of localization studies.

• Co-expression considerations: In experimental systems, researchers should determine the endogenous expression of all CELSR family members. Unrecognized CELSR2 expression could influence CELSR1 behavior through heterophilic interactions, potentially altering localization, mobility, or function.

• Functional redundancy analysis: When studying CELSR1 function using knockdown/knockout approaches, compensatory effects through heterophilic interactions with CELSR2 might mask phenotypes. Combined knockdown experiments may be necessary to reveal full functional dependencies.

• Heterophilic complex dynamics: CELSR1-CELSR2 heterophilic interactions are disrupted by EGTA , confirming their calcium dependence like homophilic interactions. Researchers should include calcium chelation controls to distinguish adhesion-dependent versus independent functions.

To address these complexities, experiments using CELSR1-FITC antibody should incorporate appropriate controls for CELSR2 expression and, when possible, include dual labeling approaches to differentiate heterophilic from homophilic complexes.

How can researchers use CELSR1-FITC antibody to investigate intersections between planar cell polarity and other developmental pathways?

Researchers can leverage CELSR1-FITC antibody to explore connections between planar cell polarity and other developmental pathways through several sophisticated approaches:

• Co-localization with pathway components: Perform multi-color immunofluorescence studies combining CELSR1-FITC antibody with markers of Wnt/β-catenin signaling, Hedgehog pathway, Notch signaling, or cytoskeletal regulators to identify potential points of pathway convergence at cellular junctions.

• Temporal correlation studies: Track CELSR1 asymmetric localization dynamics relative to activation of other pathways during developmental processes using timed sample collection and simultaneous staining for CELSR1 and phosphorylated downstream effectors of interconnected pathways.

• Perturbation analysis: Apply specific pathway modulators (small molecule inhibitors, pathway-specific siRNAs) and assess how these interventions affect CELSR1 distribution patterns or stability of PCP complexes using the FITC-conjugated antibody to quantify changes.

• Context-dependent localization: Compare CELSR1 distribution in tissues where different pathways predominate (e.g., neural tissues versus epithelial structures) to identify tissue-specific patterns of cross-regulation.

• Correlated functional analysis: Combine CELSR1 localization studies with functional readouts of multiple pathways (reporter assays, target gene expression) to establish whether physical associations correlate with functional interactions.

This integrative approach can reveal how CELSR1-mediated adhesion and PCP organization coordinate with or influence other developmental signaling networks, providing insights into the complex regulatory networks governing tissue morphogenesis.

What technical considerations should be addressed when designing experiments to study CELSR1 interactions in primary cell cultures versus established cell lines?

When studying CELSR1 interactions in primary cells versus established cell lines, researchers must address several critical technical considerations:

• Expression level variations: Primary cells often express endogenous CELSR1 at lower levels than overexpression systems. Adjust antibody dilutions accordingly (potentially using 1:50 rather than 1:200) and consider signal amplification methods for primary cells while avoiding saturation in overexpression systems.

• Cell junction maturity: Primary epithelial cells form physiologically relevant junctions that mature over time, affecting CELSR1 localization. Allow sufficient time for junction formation (typically 48-72 hours post-confluence) before antibody application, and standardize this timing across experiments.

• Fixation optimization: Primary cells may require gentler fixation protocols to preserve CELSR1 epitopes. Test different fixatives (2-4% PFA, methanol, or combined protocols) specifically optimized for each cell type rather than applying standard protocols developed for cell lines.

• Background autofluorescence: Primary cells often exhibit higher autofluorescence than established lines. Include unstained controls and consider using confocal microscopy with spectral unmixing to distinguish specific FITC signal from autofluorescence.

• Heterogeneity management: Primary cultures show greater cell-to-cell variability. Increase sampling (analyze more cells/fields) and consider using markers to identify specific cell subtypes within heterogeneous populations.

• Calcium sensitivity: Since CELSR1 adhesion is calcium-dependent , primary cells may respond differently to calcium manipulation than cell lines. When performing calcium switch experiments, optimize calcium concentrations and equilibration times specifically for each cell type.

By addressing these considerations, researchers can develop robust protocols for comparing CELSR1 biology between physiologically relevant primary cells and more experimentally tractable cell lines.

How can genetic variation in CELSR1 impact experimental outcomes in cross-species or translational research applications?

Genetic variation in CELSR1 can significantly impact experimental outcomes in cross-species and translational research through several mechanisms:

• Epitope conservation analysis: Despite the CELSR1-FITC antibody showing reactivity across human, mouse, and rat samples , sequence variations in the target epitope region (amino acids 751-850) may affect binding affinity between species. Before cross-species comparisons, researchers should align CELSR1 sequences to verify epitope conservation and validate antibody performance in each species.

• Functional domain variations: While core functions are conserved, species-specific variations in CELSR1 domains might affect interaction strength or specificity with binding partners. When translating findings between models, researchers should consider how these variations might influence interpretation of localization patterns or protein complex formation.

• Expression pattern differences: CELSR1 expression timing and tissue distribution may vary between species. When designing translational studies, preliminary mapping of expression patterns across species using the FITC-conjugated antibody can prevent misinterpretation of negative results.

• Human genetic diversity: Human populations harbor numerous CELSR1 variants of unknown significance. In translational applications, researchers should consider how common polymorphisms might affect antibody binding or protein function, potentially requiring genotyping of samples to stratify results.

• Interaction partner compatibility: When studying CELSR1 in heterologous systems (e.g., human CELSR1 in mouse cells), compatibility with species-specific interaction partners should be verified, as incomplete conservation might affect localization or function.

By accounting for these variations, researchers can develop experimental designs that appropriately control for species-specific differences, enhancing the translational relevance of CELSR1 studies across model systems.