CDH7 Antibody

What is CDH7 protein and what biological roles does it play?

CDH7, also known as cadherin-7, is a cell adhesion molecule that plays crucial roles in cell-cell adhesion and tissue organization. It functions as a key player in developmental processes and has been implicated in disease mechanisms. Its involvement in cell signaling and migration pathways suggests potential roles in cancer metastasis and tissue remodeling. CDH7 is particularly important in developmental contexts, where it contributes to proper cellular organization and differentiation . In mammalian brain development, CHD7 is predominantly expressed in cerebellar granule cells, with expression levels varying during different stages of cell differentiation . It also has significant functions in cardiovascular development, particularly in the regulation of cardiac neural crest cells (cNCCs) .

What types of CDH7 antibodies are available for research and what are their characteristics?

CDH7 antibodies are available in both polyclonal and monoclonal formats. Polyclonal antibodies like PACO57300 are produced in rabbits using recombinant human Cadherin-7 protein (typically regions such as 146-296AA) as immunogens. These antibodies typically have high reactivity with human samples and are purified using protein G, resulting in >95% purity. They are usually supplied in liquid form with storage buffers containing preservatives (e.g., 0.03% Proclin 300) and stabilizers (e.g., 50% Glycerol in 0.01M PBS, pH 7.4). The typical applications for these antibodies include Western blot (WB), immunohistochemistry (IHC), immunofluorescence (IF), and ELISA, with specific recommended dilutions for each application .

What is the difference between CDH7 antibodies and CHD7 antibodies in research contexts?

This is a critical distinction that researchers must understand. CDH7 (Cadherin-7) and CHD7 (Chromodomain Helicase DNA-binding protein 7) are entirely different proteins with distinct functions. CDH7 is a calcium-dependent cell adhesion protein involved in cell-cell interactions , while CHD7 is a chromatin remodeler that regulates gene expression through ATP-dependent mechanisms . CHD7 mutations are associated with CHARGE syndrome and affect developmental processes through regulation of enhancer activity. The antibodies targeting these proteins recognize completely different epitopes and are used to study different biological processes. Researchers must verify which protein they are actually investigating to avoid experimental confusion .

How should CDH7 antibodies be validated before use in experimental procedures?

Proper validation of CDH7 antibodies is essential for experimental reliability. Start with Western blot analysis using positive control lysates from cells known to express CDH7, such as HepG2, 293, or K562 cell lines. The antibody should detect a band at the predicted molecular weight (approximately 88 kDa for CDH7). Validation should also include negative controls, either through siRNA knockdown of CDH7 or using cell lines that don't express the protein. For immunohistochemistry or immunofluorescence applications, parallel staining with multiple antibodies recognizing different epitopes of CDH7 provides additional validation. Additionally, comparing the antibody reactivity pattern with known mRNA expression patterns (e.g., through in situ hybridization data) can further confirm specificity. Always document the validation process including antibody concentrations, exposure times, and detection methods to ensure reproducibility .

What are the optimal protocols for using CDH7 antibodies in Western blot applications?

For Western blot applications using CDH7 antibodies, optimal results are typically achieved with the following protocol: Prepare protein lysates from cells of interest (e.g., HepG2, 293, or K562 cells) using standard lysis buffers containing protease inhibitors. Load 20-50 μg of protein per lane and separate by SDS-PAGE using 8-10% gels (appropriate for the 88 kDa CDH7 protein). Transfer proteins to PVDF or nitrocellulose membranes using standard wet or semi-dry transfer systems. Block membranes with 5% non-fat milk or BSA in TBST for 1 hour at room temperature. Incubate with CDH7 primary antibody at a dilution of 1:500-1:5000 (optimally around 1:2000, or approximately 5.5 μg/ml) overnight at 4°C. After washing, incubate with appropriate HRP-conjugated secondary antibody (e.g., goat anti-rabbit IgG at 1:50000 dilution). Develop using chemiluminescence detection. The predicted band size for CDH7 is 88 kDa, which should align with observed bands in positive control samples .

How can CDH7 antibodies be effectively used in immunofluorescence applications?

For effective immunofluorescence applications with CDH7 antibodies, follow this methodological approach: Fix cells or tissue sections using 4% paraformaldehyde for 15-20 minutes at room temperature. For tissue sections, perform antigen retrieval if necessary (typically heat-mediated retrieval in citrate buffer pH 6.0). Permeabilize with 0.1-0.3% Triton X-100 in PBS for 10 minutes. Block non-specific binding with 5-10% normal serum (from the species of secondary antibody) in PBS containing 0.1% Triton X-100 for 1 hour. Incubate with CDH7 primary antibody at a dilution of 1:50-1:200 overnight at 4°C. Following thorough washing, incubate with fluorophore-conjugated secondary antibody at 1:500-1:1000 dilution for 1-2 hours at room temperature in the dark. Counterstain nuclei with DAPI and mount with anti-fade mounting medium. To confirm specificity, always include controls such as secondary-only staining and, ideally, samples known to lack CDH7 expression. Co-staining with other markers can provide valuable context, particularly in developmental studies where CDH7 expression patterns may correlate with specific cell types or differentiation states .

How does CHD7 regulate gene expression in neural crest and cardiovascular development?

CHD7 functions as a chromatin remodeler that fine-tunes gene expression rather than acting as a simple on/off switch for genes. In neural crest and cardiovascular development, CHD7 preferentially localizes to active enhancer or promoter regions marked by H3K27ac histone modifications. ChIP-seq analysis has revealed that approximately 75% of CHD7-associated genomic peaks overlap with H3K27ac marks, indicating CHD7's preference for active regulatory regions. In cardiac neural crest cells (cNCCs), deletion of Chd7 leads to significant but moderate (typically within 2.5-fold) changes in the expression of hundreds of genes, including those critical for neural crest development such as Hand2, Foxc2, and Sema3C. This supports CHD7's role as a fine-tuning regulator of developmental gene networks. The genome-wide distribution of CHD7 shows that about 5,418 CHD7-peaks are located within 20 kb of transcription start sites, affecting approximately 5,144 genes, though only a subset of these show significantly altered expression upon Chd7 deletion. This selective regulatory pattern explains why CHD7 mutations lead to specific developmental defects rather than broad developmental failures .

What are the considerations for studying CDH7/CHD7 in mouse models?

When designing experiments involving CDH7/CHD7 in mouse models, researchers should consider several critical factors. For CHD7, conditional knockout approaches using tissue-specific Cre drivers (such as Wnt1-Cre2 for neural crest cells or Atoh1-Cre for cerebellar cells) are often necessary since complete knockout leads to embryonic lethality. Reporter alleles (like R26mTmG) that express membrane-tethered GFP in CRE-recombinase cells are valuable for distinguishing recombined cells from non-recombined cells. Timing is crucial - for example, Chd7 deletion in neural crest cells becomes efficient around E10.5. Phenotypic analysis should be comprehensive, including developmental timing, morphological assessments, histological analysis, and molecular profiling through techniques like RNA-seq and ChIP-seq. For gene expression studies, consider the relatively moderate impact of Chd7 deletion (most genes change by less than 2.5-fold), which may require more sensitive detection methods and appropriate statistical approaches. Also, account for potential variability between samples even with identical genetic modifications, as similar variability is observed in CHARGE syndrome patients with identical mutations. Finally, when analyzing direct targets, combining transcriptome data with ChIP-seq can help distinguish direct from indirect effects of CHD7 activity .

How can researchers discern between direct and indirect targets of CHD7 in developmental contexts?

To differentiate between direct and indirect targets of CHD7 in developmental contexts, researchers should implement an integrated experimental approach. First, conduct CHD7 ChIP-seq in the specific cell type or tissue of interest to identify genomic regions directly bound by CHD7. In parallel, perform H3K27ac ChIP-seq to map active enhancers and promoters, as CHD7 preferentially associates with these regions. Then, generate transcriptome data (RNA-seq) from both wild-type and Chd7-deficient samples of the same cell type to identify differentially expressed genes. The integration of these datasets allows for the identification of direct targets - genes that show both CHD7 binding within their regulatory regions and altered expression upon Chd7 deletion. Based on previous studies, only a subset of CHD7-bound genes (approximately 43 out of 5,144 in one study) show significant expression changes when CHD7 is deleted, highlighting the importance of this integrated approach. To validate direct regulation, researchers can perform functional assays such as luciferase reporter assays using the identified CHD7-binding regions, or targeted deletion of these regions using CRISPR-Cas9 to confirm their role in gene regulation. Additionally, assessing the kinetics of expression changes after inducible Chd7 deletion can help distinguish immediate (likely direct) from delayed (potentially indirect) effects .

What experimental designs are most effective for studying CDH7 in cancer progression and metastasis?

For studying CDH7's role in cancer progression and metastasis, a multi-faceted experimental approach is recommended. Begin with expression analysis across cancer types and stages using tissue microarrays and public databases to identify cancer types where CDH7 shows altered expression. For functional studies, establish stable cell lines with CDH7 overexpression, knockdown, or knockout using lentiviral or CRISPR-Cas9 systems. These modified cells should be characterized for changes in adhesion properties, migration capacity (using wound healing and transwell assays), invasion potential (using Matrigel invasion assays), and proliferation rates. For in vivo metastasis studies, orthotopic implantation models are preferable to subcutaneous xenografts as they better recapitulate the natural tumor microenvironment. Tracking metastasis can be facilitated by using luciferase or fluorescent protein-tagged cells for in vivo imaging. At the molecular level, examine CDH7's interaction partners through co-immunoprecipitation followed by mass spectrometry, and investigate downstream signaling pathways affected by CDH7 modulation. Additionally, consider patient-derived xenograft (PDX) models for more clinically relevant insights, and correlate findings with patient outcome data to establish clinical significance. Throughout these studies, using validated CDH7 antibodies for detection and quantification is essential, with appropriate controls to ensure specificity .

What are common issues with CDH7 antibodies in experimental applications and how can they be resolved?

Researchers frequently encounter several challenges when working with CDH7 antibodies. One common issue is non-specific binding in Western blots, which can be addressed by optimizing blocking conditions (trying different blockers like 5% milk, 3-5% BSA, or commercial blockers) and increasing the stringency of wash steps (adding more Tween-20 or salt to wash buffers). For weak or absent signals, consider optimizing antibody concentration, extending incubation times, or using more sensitive detection systems. Some antibodies may require specific antigen retrieval methods for IHC/IF applications—try different retrieval buffers (citrate pH 6.0, EDTA pH 8.0, or Tris-EDTA pH 9.0) and methods (microwave, pressure cooker, or water bath). Background issues in immunofluorescence can often be resolved by extending blocking times, using stronger detergents during permeabilization, or implementing additional blocking steps with normal serum. For all applications, batch-to-batch variation can be problematic; always validate new lots against previously successful lots using positive control samples. Finally, some epitopes may be masked by protein-protein interactions or post-translational modifications—consider denaturing conditions for Western blot and appropriate fixation methods for IHC/IF to expose relevant epitopes .

How can researchers distinguish between specific and non-specific signals when using CDH7 antibodies?

Distinguishing between specific and non-specific signals requires implementing multiple validation strategies. First, always include positive controls (cells or tissues known to express CDH7) and negative controls (cells with CDH7 knockdown/knockout or tissues known not to express CDH7). For Western blots, specific signals should appear at the predicted molecular weight (88 kDa for CDH7), while non-specific bands will appear at unexpected sizes. Pre-absorption tests can be valuable—pre-incubate the antibody with excess purified antigen (if available), which should eliminate specific signals but leave non-specific binding intact. For immunostaining applications, compare staining patterns with in situ hybridization data or RNA-seq expression data to confirm that protein detection aligns with mRNA expression patterns. Using two different antibodies targeting distinct epitopes of CDH7 can provide confirmation—consistent signals from both antibodies strongly indicate specificity. Additionally, proper experimental controls should be included in each experiment: secondary-only controls to assess background from the secondary antibody, isotype controls to evaluate non-specific binding of the primary antibody class, and system-specific controls (such as cells transfected with CDH7 expression constructs versus empty vectors). Finally, comparing results across different detection methods (e.g., WB, IHC, and IF) can provide further validation of specificity .

How should researchers approach experimental design when studying potential interactions between CDH7 and CHD7 functions?

When investigating potential functional interactions between the cell adhesion molecule CDH7 and the chromatin remodeler CHD7, researchers should implement a carefully structured experimental approach. First, establish clear expression profiles for both proteins across relevant cell types and developmental stages using validated antibodies and qRT-PCR. Co-expression analysis can identify contexts where both proteins are present simultaneously. For potential regulatory relationships, determine if CHD7 binds to regulatory regions of the CDH7 gene through CHD7 ChIP-seq, and assess whether CDH7 expression is altered in CHD7-deficient models. Conversely, investigate whether CDH7-mediated adhesion affects nuclear signaling that might influence CHD7 activity. For functional studies, develop systems where both proteins can be independently manipulated (knockdown, knockout, overexpression) in relevant cell types, using inducible systems to control timing. Phenotypic analyses should focus on processes potentially regulated by both proteins, such as cell migration, differentiation, or tissue morphogenesis. Co-immunoprecipitation experiments can assess whether the proteins exist in the same complexes, though direct interaction is unlikely given their different subcellular localizations. Finally, examine downstream effects of manipulating each protein individually and simultaneously to identify synergistic, additive, or antagonistic relationships. Throughout this research, careful labeling and documentation are crucial to avoid confusion between these similarly named but functionally distinct proteins .

What are the recommended dilutions and applications for CDH7 antibodies in different experimental protocols?

Note: These values are based on the PACO57300 CDH7 antibody and may require optimization for specific experimental conditions and sample types .

What is the tissue-specific expression pattern of CHD7 during cerebellar development?

Note: Expression levels were determined through immunostaining, in situ hybridization, and reporter gene expression using Chd7-GFP mice .

What direct target genes does CHD7 regulate in neural development?

Note: These findings are based on transcriptome analysis of CHD7-deficient cells and CHD7 ChIP-seq experiments in neural crest-derived tissues .