CDH12 Antibody

What is CDH12 and what are its primary biological functions?

CDH12 (also known as brain-cadherin) is a subtype of neural cadherin first identified in the brain but later found in various tissues. It plays crucial roles in neurite outgrowth through the PKA/Rac1 pathway, influencing neuronal development and axonal extension. Research has demonstrated that CDH12 affects Rac1/Cdc42 phosphorylation through PKA-dependent mechanisms, which directly impacts neuronal development . Beyond the nervous system, CDH12 has been implicated in cancer cell proliferation and metastasis, particularly in colorectal cancer where it promotes epithelial-mesenchymal transition (EMT) by targeting the transcriptional factor Snail .

Which tissues express CDH12 during development and in adult organisms?

CDH12 exhibits distinct expression patterns across developmental stages and tissues. It shows high expression in human embryonic tissues, with particularly strong presence in retinal pigmented epithelium followed by developing kidneys . Immunohistochemical analyses reveal CDH12 expression in fetal kidney structures including the ureteric bud (precursor of collecting ducts), early condensates where epithelialization begins, and developing proximal tubules at 12-15 weeks post-conception . In adult tissues, CDH12 maintains high expression in the brain but is notably absent in adult kidneys, suggesting a developmental-specific role in kidney formation . Transcriptomic analyses of human embryonic tissues (33-55 days post-conception) confirm widespread expression during organogenesis .

What are the optimal conditions for CDH12 antibody storage and handling?

For optimal antibody performance, CDH12 antibodies should be stored according to manufacturer specifications, typically at -20°C for long-term storage. When working with CDH12 antibodies for cell-based applications, it's important to note that classic cadherins can be protected from trypsin treatment in the presence of Ca²⁺, which affects cell harvesting protocols . For immunostaining applications, researchers should maintain consistent storage conditions as the analyte (CDH12) does not exhibit high stability under variable conditions, which can impact detection sensitivity and reproducibility . Freeze-thaw cycles should be minimized, and working aliquots prepared to maintain antibody integrity during experimental procedures.

What are the recommended protocols for CDH12 detection in tissue samples?

For immunohistochemical detection of CDH12 in tissue samples, published protocols indicate effective staining using anti-CDH12 antibodies at a dilution of 1:100 (e.g., ab71055 from Abcam) . The tissue preparation typically involves fixation, sectioning, and antigen retrieval steps followed by blocking with bovine serum albumin (10 mg/mL) containing 10% normal goat serum at 37°C for 2 hours . For cellular samples, washing with PBS three to five times after fixation is recommended before proceeding with primary antibody incubation at 4°C overnight . Visualization can be achieved using appropriate secondary antibodies conjugated to fluorophores for immunofluorescence applications, with controls to verify specificity.

What dilution ranges work best for CDH12 antibodies in different applications?

The optimal dilution of CDH12 antibodies varies by application and specific antibody used. For immunohistochemistry, dilutions around 1:100 have been successfully employed . In immunofluorescence studies of neuronal cultures, anti-CDH12 antibodies have been used at dilutions of 1:100 to 1:500 . For Western blot applications, researchers commonly use dilutions in the range of 1:500 to 1:1000, though optimization is recommended for each specific antibody and experimental system. When designing experiments, validation studies with positive and negative controls should be conducted to determine the optimal working dilution for the specific antibody and application.

How can researchers quantitatively analyze CDH12 levels in biological fluids?

Surface Plasmon Resonance imaging (SPRi) testing provides a sophisticated approach for quantitative detection of CDH12 in human plasma and peritoneal fluid. The method has demonstrated high sensitivity with a Limit of Quantification (LOQ) of 4.92 pg mL⁻¹, allowing direct quantitative analysis without sample concentration . This technique relies on the thermodynamic stability of the anti-CDH12 antibody-CDH12 protein complex, characterized by an association equilibrium constant (KA = 1.66 × 10¹¹ dm³ mol⁻¹) and dissociation equilibrium constant (KD = 7.52 × 10⁻¹² mol dm⁻³) . The optimal antibody concentration for biosensor applications is 15 ng mL⁻¹, as concentrations above this value do not increase covalent binding to the biosensor surface .

How can CDH12 knockdown or overexpression be effectively achieved in neuronal models?

For CDH12 knockdown experiments, RNA interference using short hairpin RNA (shRNA) targeting CDH12 has been successfully employed. GFP-labeled shRNA constructs can be transfected into neuronal cells using Lipofectamine™ 2000 when cells reach approximately 70% confluence . Effective targeting sequences include: shCDH12-1: 5′-CACCGCTGGGCAACAATTCTCCTTTTCAAGAGAAGGAGAATTGTTGCCCAGCTTTTTG-3′ (sense sequence) . After transfection, cells should be harvested at 48 hours to assess knockdown efficiency via Western blot, and stable expression clones can be selected using G418 antibiotics . For overexpression studies, expression vectors containing the CDH12 coding sequence can be transfected following similar protocols. Functional validation of successful genetic manipulation can be performed through neurite outgrowth assays, as CDH12 has been shown to regulate axon extension in E18 neurons .

What experimental approaches can be used to investigate CDH12's role in epithelial-mesenchymal transition (EMT)?

To study CDH12's role in EMT, researchers can employ multiple complementary approaches. First, modulating CDH12 expression (through knockdown or overexpression) in epithelial cancer cell lines allows assessment of changes in EMT markers. Western blot analysis should target EMT regulators like Snail, E-cadherin, N-cadherin, and vimentin to determine how CDH12 affects their expression patterns . Cell migration and invasion capability can be evaluated using transwell assays, with CDH12 manipulated cells showing altered invasive properties correlating with EMT status . For mechanistic studies, investigating the relationship between CDH12 and MCP1 (monocyte chemotactic protein 1) is recommended, as research indicates MCP1 promotes CDH12 expression through induction of MCP1-induced protein (MCPIP) . Additionally, researchers should analyze the expression of CDH12 in patient tumor samples and correlate with invasive capability and clinical parameters to establish clinical relevance.

How can researchers address potential cross-reactivity issues with CDH12 antibodies?

Addressing cross-reactivity is crucial for CDH12 antibody application validity. First, researchers should conduct comprehensive specificity validation using positive controls (tissues known to express CDH12, such as brain tissue) and negative controls (adult kidney tissue, which lacks CDH12 expression) . Western blot analysis can confirm antibody specificity by verifying detection of a single band at the expected molecular weight. For complex tissue samples, pre-absorption controls with recombinant CDH12 protein can be used to confirm binding specificity. Additionally, comparing results using multiple antibodies targeting different CDH12 epitopes can increase confidence in specificity. The selectivity of CDH12 detection methods has been validated against potential interferents occurring at up to 100-fold excess concentration relative to CDH12, confirming methodological robustness .

How is CDH12 involved in cancer progression, and what methodologies best assess this relationship?

CDH12 has demonstrated significant associations with cancer progression, particularly in colorectal cancer (CRC). Immunohistochemical studies have shown higher CDH12 expression in tumor tissues compared to adjacent normal tissues, with positive staining primarily localized along the cell membrane . To methodologically assess this relationship, researchers should employ tissue microarrays with paired tumor and normal samples, evaluating CDH12 expression patterns (uniform positive, heterogeneous, or negative) and correlating with clinicopathological parameters. Statistical analyses have revealed that CDH12 expression correlates significantly with tumor invasion depth (p = 0.02) and lymph node metastasis (p = 0.04) .

For functional studies, cell viability assays (e.g., CCK-8) comparing CDH12-knockdown and control cells can quantify proliferation effects, while transwell assays assess migration and invasion capabilities . Kaplan-Meier survival analysis is recommended to evaluate CDH12 as a prognostic marker, as high expression predicts poorer outcomes in CRC patients .

What techniques can effectively visualize and quantify CDH12's role in neurite outgrowth?

To investigate CDH12's role in neurite outgrowth, researchers can employ a combination of molecular and imaging techniques. RNA interference targeting CDH12 in primary neuronal cultures (particularly E18 neurons) allows observation of resulting changes in axon extension . Immunofluorescence staining using anti-Tuj1 antibody (1:1000) alongside CDH12 antibody (1:100) enables visualization of neuronal structures and CDH12 localization . Quantitative analysis should include measurements of axon length, branching complexity, and growth cone area using appropriate imaging software.

For deeper mechanistic insights, researchers can probe the PKA/Rac1 pathway by employing PKA inhibitors (H-89) or activators (8-Bromo-cAMP sodium salt) in combination with CDH12 manipulation, followed by assessment of Rac1/Cdc42 phosphorylation status . Transcriptome profiling of neurons with or without CDH12 knockdown provides comprehensive pathway analysis revealing molecular mechanisms underlying CDH12's effects on neurite outgrowth .

How can CDH12's role in kidney development be experimentally investigated?

To study CDH12's role in kidney development, researchers should leverage its distinct expression pattern - present in fetal kidneys but absent in adult kidneys . Immunohistochemical staining of human fetal kidney sections at various developmental stages (particularly 12-15 weeks post-conception) using anti-CDH12 antibodies (1:100 dilution) can visualize expression in specific structures including the ureteric bud, early condensates, and proximal tubules . For genetic association studies, researchers can examine CDH12 variants in cohorts with congenital kidney abnormalities, such as posterior urethral valves (PUVs) or ureteropelvic junction obstruction (UPJO) .

Expression analysis comparing adult and fetal tissue samples can be performed using quantitative PCR, with adult brain tissue serving as a positive control and adult kidney as a negative control . For functional studies, CDH12 knockdown in kidney organoid models derived from human pluripotent stem cells would allow assessment of its role in nephron formation and tubular development.

What are the critical parameters for optimizing SPRi-based CDH12 detection?

For optimal SPRi-based CDH12 detection, several critical parameters must be carefully controlled. The antibody concentration applied to biosensor surfaces should be maintained at 15 ng mL⁻¹, as this has been determined to be the saturation point for covalent binding . The pH of testing solutions should be standardized at 7.40 to ensure consistent antibody-antigen interactions . The thermodynamic parameters of the anti-CDH12 antibody-CDH12 complex (KA = 1.66 × 10¹¹ dm³ mol⁻¹ and KD = 7.52 × 10⁻¹² mol dm⁻³) indicate a highly stable interaction that can be leveraged for sensitive detection .

For accurate quantification, researchers should prepare standard curves using recombinant CDH12 protein in the concentration range of 20-100 ng mL⁻¹, as these levels have been validated for reliable constant determination . Sample storage conditions are particularly critical, as CDH12 has limited stability; therefore, consistent storage protocols must be established to preserve analyte integrity for reproducible results .

What controls are essential when studying CDH12 expression across different tissues and developmental stages?

When studying CDH12 expression across tissues and developmental stages, several controls are essential for accurate interpretation. Positive controls should include brain tissue, which consistently shows high CDH12 expression in both developing and adult stages . Adult kidney tissue serves as an excellent negative control, as CDH12 expression is absent in mature kidneys despite being present during development . For developmental studies, temporal controls comparing tissues at different gestational ages (e.g., 12 vs. 15 weeks post-conception) help track expression dynamics .

Technical controls should include secondary antibody-only samples to detect non-specific binding, isotype controls to verify primary antibody specificity, and RNA-level validation (qPCR) to complement protein-level findings . When comparing expression across tissues, housekeeping genes or proteins that maintain stable expression regardless of tissue type or developmental stage should be used for normalization, and consistent image acquisition settings must be maintained for valid comparisons.

How can contradictory findings in CDH12 functional studies be reconciled through experimental design?

Reconciling contradictory findings in CDH12 functional studies requires careful experimental design considerations. First, researchers should examine model system differences, as CDH12's functions may vary between in vitro and in vivo systems or across different cell types. For instance, while CDH12 promotes neurite outgrowth in primary neurons , it may exhibit different effects in established cell lines. Comprehensive documentation of experimental conditions is crucial, including cell culture parameters, transfection efficiency, protein expression levels, and functional readouts.

For knockdown experiments, using multiple shRNA sequences targeting different regions of CDH12 helps validate that observed effects are specifically due to CDH12 depletion rather than off-target effects . Rescue experiments, where wild-type CDH12 is reintroduced into knockdown cells, provide strong evidence for functional specificity. Additionally, time-course experiments can reveal whether seemingly contradictory outcomes represent different temporal phases of the same process. Finally, investigating context-dependent effects through systematic manipulation of related signaling pathways (e.g., PKA/Rac1 or MCP1/MCPIP in CDH12-relevant contexts) can help identify the conditions under which CDH12 functions differently .