CELSR2 Antibody

What is CELSR2 and why is it significant for research?

CELSR2 (Cadherin EGF LAG seven-pass G-type receptor 2) is a 7-transmembrane helix receptor that contains nine cadherin-like domains, seven EGF-like repeats, and 2 laminin A G-type repeats . This protein plays a crucial role in cell/cell signaling during nervous system formation and is also involved in planar cell polarity (PCP) and Wnt signaling pathways, where it interacts with key proteins such as Frizzled and Dishevelled .

The protein is also known by several alternative names including EGFL2, ADGRC2, CDHF10, Flamingo1, EGF-like protein 2, and EGF-like-domain multiple 2 . In terms of structure, CELSR2 is a large protein with a molecular weight of approximately 317.5 kilodaltons .

Research significance:

• Essential component in developmental biology studies

• Implicated in several signaling pathways critical for cell polarity

• Emerging biomarker in cancer research, particularly hepatocellular carcinoma

• Potential therapeutic target due to its role in cancer progression

What experimental applications are CELSR2 antibodies optimized for?

CELSR2 antibodies have been validated for multiple experimental techniques with varying recommended dilutions. Based on comprehensive analysis of commercially available antibodies, the following applications and typical working dilutions are recommended:

When selecting applications, researchers should consider that CELSR2 subcellular localization is predominantly cytosolic in multiple cell lines, including A-431 and U-251 MG .

How should researchers validate CELSR2 antibody specificity?

A comprehensive validation strategy for CELSR2 antibodies should include:

• Positive control selection: Use tissues/cells known to express CELSR2, such as:

  • SH-SY5Y neuroblastoma cells show consistent detection

  • Brain tissue exhibits high endogenous expression

  • HEK293 cells transfected with human CELSR2

• Negative controls implementation:

  • Use PBS instead of primary antibody as technical negative control

  • Include tissues with minimal CELSR2 expression (based on HPA data, normal liver tissue shows relatively low expression)

• Size verification: Confirm the ~317-320 kDa band in Western blot, though some antibodies may detect cleavage products (e.g., 72 kDa observed band with calculated MW of 317 kDa)

• Immunogen compatibility assessment: Verify that antibodies targeting different epitopes (N-terminal vs C-terminal) provide consistent results

• Genetic manipulation verification: Knockdown or knockout experiments to confirm signal specificity (depletion of CELSR2 should result in reduced antibody signal)

How can CELSR2 antibodies be utilized to investigate CELSR2's role in cancer progression?

CELSR2 has emerged as a potential biomarker and therapeutic target in cancer research, particularly in hepatocellular carcinoma (HCC). Studies have demonstrated that CELSR2 mRNA and protein expression levels are significantly higher in cancerous tissue than in normal tissue, and increased expression correlates with poor prognosis .

Methodological approach for cancer studies:

• Expression profiling across cancer stages:

  • IHC analysis of tissue microarrays containing different tumor stages

  • Correlation with clinicopathological parameters as shown in recent studies where CELSR2 expression was positively correlated with tumor stage in HCC patients

• Survival analysis methodology:

  • Categorize samples into high and low CELSR2 expression groups

  • Apply Kaplan-Meier analysis with hazard ratio calculation (recent data showed HR = 1.43, 95% CI: 1.01–2.03, p = 0.042 for HCC patients)

• Functional studies:

  • RNA interference or CRISPR-based knockdown/knockout of CELSR2 in cancer cell lines

  • Analysis of phenotypic changes (proliferation, invasion, etc.) as demonstrated in liver cancer research where CELSR2 depletion inhibited proliferation and invasion

• Molecular mechanistic investigation:

  • Co-immunoprecipitation with CELSR2 antibodies to identify binding partners

  • Pathway analysis to elucidate downstream signaling events

What technical challenges exist when detecting CELSR2 protein, and how can they be overcome?

CELSR2's large molecular weight (317-320 kDa) and transmembrane nature present several technical challenges:

• Protein extraction optimization:

  • Use specialized lysis buffers containing adequate detergents (e.g., 1% Triton X-100 or RIPA buffer)

  • Include protease inhibitors to prevent degradation

  • Consider mechanical disruption methods for complete membrane protein solubilization

• Western blot optimization for high molecular weight proteins:

  • Use low percentage gels (6-7%) or gradient gels

  • Extend transfer time (overnight at low voltage) with larger pore size membranes

  • Include SDS in transfer buffer to facilitate movement of large proteins

  • Consider wet transfer instead of semi-dry methods

• Epitope accessibility challenges in fixed tissues:

  • For IHC applications, optimized antigen retrieval is critical

  • Recommended: TE buffer at pH 9.0 as an alternative to standard citrate buffer (pH 6.0)

  • Extended primary antibody incubation (overnight at 4°C) improves detection

• Signal amplification strategies:

  • Consider tyramide signal amplification for weakly expressed samples

  • Use high-sensitivity detection systems (e.g., SuperSignal West Femto for WB)

  • For IHC/IF, biotin-streptavidin systems can enhance signal

How can researchers effectively design experiments to investigate CELSR2 genetic alterations?

CELSR2 genetic alterations occur in approximately 8% of HCC patients, including mRNA overexpression (4.01%), mutations (3.15%), amplifications (0.29%), and multiple alterations (0.57%) . To effectively study these alterations:

• Mutation profiling strategy:

  • Target sequencing of CELSR2 hotspots

  • Database mining from TCGA and other cancer genomics resources

  • Focus on missense mutations, which represent the most common type of CELSR2 alterations

• Expression quantification approaches:

  • qRT-PCR for mRNA level assessment

  • Western blot and IHC for protein-level validation

  • Correlation of expression with genetic alterations

• Functional characterization of mutations:

  • Site-directed mutagenesis to introduce specific mutations

  • Stable cell lines expressing mutant CELSR2 variants

  • Phenotypic and signaling pathway assessment

• Co-expressed gene analysis:

  • Utilize resources like LinkedOmics to identify genes co-expressed with CELSR2

  • Perform pathway enrichment analysis to understand biological implications

  • Example: The correlation between CELSR2 and its co-expressed genes in HCC can be analyzed through GO and KEGG pathway analysis

What are the specific considerations for studying CELSR2 in different tissue and cell types?

CELSR2 expression varies significantly across tissues and cell types, necessitating tailored experimental approaches:

• Tissue-specific expression patterns:

  • Brain tissue shows highest expression and requires special handling protocols

  • Liver tissue shows relatively low expression in normal conditions but increased expression in HCC

  • Breast tissue shows localization to ductal epithelium

• Cell line selection guidance:

  • Neuronal lines (SH-SY5Y) commonly used as positive controls

  • Endothelial cells (bEnd.3) express detectable CELSR2 levels

  • HEK293 cells serve as good transfection models for overexpression studies

• Species cross-reactivity considerations:

  • Most antibodies react with human CELSR2

  • Many also cross-react with mouse and rat orthologs

  • Some have broader reactivity including bovine, canine, porcine, and monkey samples

• Application-specific optimizations:

  • For neuronal studies, consider co-staining with neuronal markers

  • For cancer research, pair with cell cycle markers

  • For developmental studies, embryonic tissue requires careful fixation and handling

What are the most common technical issues when working with CELSR2 antibodies and their solutions?

How do researchers optimize antibody dilutions for maximum specificity and sensitivity?

A methodical titration approach is essential for achieving optimal specificity and sensitivity:

• Initial broad range titration:

  • For IHC/IF: Begin with manufacturer's recommendation, typically between 1:50-1:500

  • For WB: Start with 1:500-1:1000 dilution range

  • For FC: Initial testing at 0.25 μg per 10^6 cells

• Sequential optimization strategy:

  • Perform a 2-fold or 3-fold dilution series around the initial dilution

  • Assess signal-to-noise ratio at each dilution

  • Document both positive signal intensity and background levels

• Application-specific considerations:

  • For IHC: Different fixatives may require different optimal dilutions

  • For IF: Consider autofluorescence of the tissue/cells being studied

  • For WB: Transfer efficiency affects optimal concentration needed

• Validation across sample types:

  • Test optimized dilution across multiple positive and negative samples

  • Verify consistent results across technical replicates

  • Consider lot-to-lot variations when using different antibody batches

What are the most reliable positive and negative controls for CELSR2 antibody validation?

Recommended positive controls:

• Tissue controls:

  • Brain tissue (particularly mouse brain) shows consistent expression

  • Human breast tissue (ductal epithelium) demonstrates specific staining

  • HCC tissue samples exhibit elevated expression compared to adjacent normal tissue

• Cell line controls:

  • SH-SY5Y human neuroblastoma cells show reliable expression

  • HEK293 cells transfected with human CELSR2

  • bEnd.3 mouse endothelioma cell line

Recommended negative controls:

• Technical negative controls:

  • Primary antibody omission (secondary antibody only)

  • Isotype control antibody (e.g., AB-108-C as used in flow cytometry validations)

  • Blocking peptide competition assay using the immunogen peptide

• Biological negative controls:

  • Tissues with minimal CELSR2 expression based on database information

  • CELSR2 knockdown/knockout cell lines or tissues

  • Cell lines known to have low CELSR2 expression

How can CELSR2 antibodies contribute to research on planar cell polarity and Wnt signaling?

CELSR2 plays a significant role in planar cell polarity (PCP) and Wnt signaling pathways, interacting with proteins such as Frizzled and Dishevelled . Advanced research approaches include:

• Protein interaction network mapping:

  • Co-immunoprecipitation with CELSR2 antibodies followed by mass spectrometry

  • Proximity ligation assays to visualize protein-protein interactions in situ

  • FRET/BRET analysis for real-time interaction dynamics

• Spatial organization analysis:

  • Super-resolution microscopy (STED, PALM, STORM) using CELSR2 antibodies

  • Co-localization studies with other PCP pathway components

  • Live-cell imaging of CELSR2 dynamics during polarization events

• Signaling pathway dissection:

  • Phospho-specific antibodies to track CELSR2 activation state

  • Quantitative analysis of downstream signaling components after CELSR2 manipulation

  • Computational modeling of CELSR2's role in PCP network dynamics

• Developmental biology applications:

  • Tracking CELSR2 expression and localization during embryonic development

  • Correlation with tissue morphogenesis and cell migration patterns

  • Integration with gene expression data to build comprehensive models

What is the potential for CELSR2 as a prognostic biomarker based on current research?

Recent studies have identified CELSR2 as a potential prognostic biomarker, particularly in hepatocellular carcinoma:

• Prognostic value evidence:

  • High CELSR2 expression significantly correlates with poor prognosis in HCC patients (HR = 1.43, 95% CI: 1.01–2.03, p = 0.042)

  • CELSR2 expression correlates positively with tumor stage and grade

  • Multivariate Cox regression analyses identified CELSR2 as an independent risk factor for HCC patients

• Implementation methodology:

  • IHC scoring systems based on staining intensity and percentage of positive cells

  • Image analysis software for quantitative assessment of CELSR2 expression

  • Integration with other molecular markers for improved prognostic accuracy

• Clinical correlation approaches:

  • Large-scale tissue microarray analysis across patient cohorts

  • Correlation with clinical parameters (tumor size, metastasis, recurrence)

  • Long-term follow-up studies to validate prognostic significance

• Multi-omics integration strategy:

  • Combine protein expression data with genomic alterations (8% alteration rate in HCC)

  • Integrate with transcriptomic profiles of co-expressed genes

  • Develop predictive algorithms incorporating multiple biomarkers

How can researchers study CELSR2's role in neurodevelopment using available antibodies?

CELSR2's high expression in brain tissue and its role in nervous system formation make it a key target for neurodevelopmental research:

• Developmental expression profiling:

  • Temporal analysis across embryonic, postnatal, and adult stages

  • Regional brain expression mapping using IHC/IF

  • Co-expression with neurodevelopmental markers

• Neuronal polarity and migration studies:

  • Live imaging of CELSR2 during neuronal polarization and axon formation

  • Analysis of CELSR2 localization in growth cones and dendritic spines

  • Microfluidic chambers to study CELSR2's role in axon guidance

• Neural circuit formation analysis:

  • CELSR2 expression patterns in developing neural circuits

  • Effects of CELSR2 manipulation on synaptogenesis and circuit assembly

  • Electrophysiological correlates of CELSR2 dysfunction

• 3D neural organoid applications:

  • CELSR2 expression dynamics during organoid development

  • Manipulation of CELSR2 function in brain organoids

  • Correlation with structural abnormalities and functional outcomes