NCAPD2 Antibody

What is NCAPD2 and what is its primary function in cellular processes?

NCAPD2 functions as a regulatory subunit of the condensin complex, required for the conversion of interphase chromatin into mitotic-like condensed chromosomes. The condensin complex introduces positive supercoils into relaxed DNA in the presence of type I topoisomerases and converts nicked DNA into positive knotted forms when type II topoisomerases are present. NCAPD2 may specifically target the condensin complex to DNA through its C-terminal domain. The protein is also known by several alternative names including CAPD2, CNAP1, and hCAP-D2 .

Beyond its primary role in chromosome condensation, NCAPD2 promotes the resolution of double-strand DNA catenanes (intertwines) between sister chromatids. Condensin-mediated compaction increases tension in catenated sister chromatids, providing directionality for type II topoisomerase-mediated strand exchanges toward chromatid decatenation. Additionally, NCAPD2 is required for the decatenation of non-centromeric ultrafine DNA bridges during anaphase .

How is NCAPD2 expression related to cancer progression?

NCAPD2 shows significantly elevated expression in multiple cancer types compared to their corresponding normal tissues. According to pan-cancer analyses, NCAPD2 is highly expressed in 25 different tumor types, including breast cancer, colon cancer, lung adenocarcinoma, and oral squamous cell carcinoma (OSCC) .

What are the commercially available NCAPD2 antibodies and their applications?

Commercial NCAPD2 antibodies are available in different formats, each optimized for specific applications:

These antibodies are primarily used in Western blotting (WB) and immunohistochemistry on paraffin-embedded tissues (IHC-P), with researchers finding NCAPD2 expression highest in proliferating cells, especially in tissues with active cell division such as bone marrow and certain epithelial tissues .

How can NCAPD2 antibodies be used to study cancer progression mechanisms?

NCAPD2 antibodies serve as essential tools for investigating its role in cancer progression through multiple experimental approaches. In breast cancer research, NCAPD2 has been shown to promote cancer progression through E2F1 and CDK1, making it a potential therapeutic target . The methodological approach involves:

• Expression Analysis: Immunohistochemical (IHC) staining to quantify NCAPD2 levels in tumor versus normal tissues. Researchers typically use scoring systems that combine staining area (scored as 0: <5%, 1: 6-25%, 2: 26-50%, 3: 51-75%, 4: 75-100%) and staining intensity (scored as 0: signal less color, 1: brown, 2: light yellow, 3: dark brown) .

• Functional Studies: shRNA-mediated NCAPD2 knockdown to investigate effects on proliferation, cloning, apoptosis, cell cycle, and migration of cancer cells. This approach has revealed that NCAPD2 downregulation inhibits OSCC cell proliferation and migration .

• Mechanistic Investigations: Techniques like chromatin immunoprecipitation (ChIP) to elucidate how NCAPD2 interacts with transcription factors such as E2F1. In MDA-MB-231 cells with NCAPD2 overexpression, ChIP experiments using E2F1 antibodies have helped identify downstream targets like CDK1 .

What is the relationship between NCAPD2 expression and immune infiltration in the tumor microenvironment?

NCAPD2 expression significantly correlates with immune cell infiltration in various tumors, suggesting its potential role in immune regulation. Comprehensive analysis using the Tumor Immune Estimation Resource (TIMER) database has demonstrated relationships between NCAPD2 expression and six types of immune cell infiltration, including:

• B cells

• CD8+ T cells

• CD4+ T cells

• Macrophages

• Neutrophils

• Dendritic cells

Additionally, NCAPD2 expression correlates with immune checkpoint expression, tumor mutation burden (TMB), microsatellite instability (MSI), and RNA methylation regulators across multiple cancer types. This correlation suggests NCAPD2 may influence immunotherapy efficiency, making it a potential biomarker for predicting response to immune checkpoint blockade treatments .

How does NCAPD2 influence neuronal development and what research methods can explore this function?

Early in neurogenesis, NCAPD2 plays an essential role in ensuring accurate mitotic chromosome condensation in neuronal stem cells, ultimately affecting neuron pool and cortex size . Research approaches to investigate this function include:

• Conditional knockout models in developing neural tissue

• Immunofluorescence microscopy with NCAPD2 antibodies to visualize expression patterns during different stages of neural development

• Live-cell imaging combined with NCAPD2 antibody staining to track chromosome condensation in dividing neural stem cells

• Correlation studies between NCAPD2 expression/mutation and neurodevelopmental disorders

Understanding NCAPD2's role in neurodevelopment requires sophisticated microscopy techniques coupled with genetic manipulation approaches and careful phenotypic analysis of resulting neural tissues.

What are the optimal conditions for Western blot analysis using NCAPD2 antibodies?

Successful Western blot analysis of NCAPD2 requires careful optimization due to its relatively large molecular weight (~150 kDa). Recommended protocol modifications include:

• Gel Preparation: Use lower percentage (6-8%) SDS-PAGE gels to properly resolve high molecular weight proteins.

• Protein Transfer: Implement longer transfer times (overnight at low voltage or 2-3 hours at higher voltage) with addition of SDS (0.1%) to the transfer buffer to facilitate movement of large proteins.

• Antibody Dilution: Primary NCAPD2 antibodies typically work optimally at 1:500 to 1:1000 dilutions, but this should be determined empirically for each specific antibody.

• Incubation Time: Extend primary antibody incubation to overnight at 4°C to improve signal strength.

• Detection Method: Enhanced chemiluminescence (ECL) with longer exposure times often yields better results for detecting NCAPD2.

For studying breast cancer cell lines such as BT549 and MDA-MB-231, researchers have successfully used these optimization strategies to detect NCAPD2 protein expression changes following genetic manipulation .

What are the best practices for immunohistochemistry (IHC) using NCAPD2 antibodies?

Optimizing IHC protocols for NCAPD2 detection in tissue samples requires attention to several critical factors:

• Antigen Retrieval: Heat-induced epitope retrieval (HIER) in citrate buffer (pH 6.0) typically provides optimal results.

• Blocking: Extended blocking (1-2 hours) with 5-10% normal serum from the species of the secondary antibody to minimize background.

• Antibody Validation: Include positive control tissues known to express NCAPD2 (bone marrow, proliferative epithelial tissues) and negative controls (antibody diluent only).

• Scoring System: Implement a standardized scoring system combining staining intensity and proportion of positively stained cells. Most researchers use a semi-quantitative approach multiplying expression intensity (0-3) by expression area (0-4) .

• Image Analysis: Multiple random fields (typically 10) should be evaluated under 200× magnification to ensure representative scoring.

This methodology has been successfully employed to demonstrate that NCAPD2 protein expression is significantly higher in colon cancer, breast cancer, lung adenocarcinoma, and lung squamous cell carcinoma compared to corresponding normal tissues .

How can researchers effectively design RNA interference experiments to study NCAPD2 function?

RNA interference (RNAi) approaches, particularly shRNA-mediated knockdown, have proven effective for studying NCAPD2 function in cancer cells. Key considerations include:

• shRNA Design: Target sequences should be specific to NCAPD2 with minimal off-target effects. Multiple shRNA constructs should be tested to identify those with highest knockdown efficiency.

• Validation: Knockdown efficiency must be validated at both mRNA level (using qPCR) and protein level (using Western blot with NCAPD2 antibodies).

• Controls: Include non-targeting shRNA controls (shCtrl) to account for non-specific effects of the viral transduction process.

• Functional Assays: Following confirmed knockdown, analyze effects on:

  • Cell proliferation (using MTT or BrdU assays)

  • Colony formation

  • Apoptosis (Annexin V/PI staining)

  • Cell cycle distribution (PI staining and flow cytometry)

  • Cell migration and invasion (Transwell assays)

• In vivo Validation: For stronger evidence, test effects of NCAPD2 knockdown in xenograft mouse models. Studies using MDA-MB-231 cells with NCAPD2 knockdown injected subcutaneously into mice have shown reduced tumor growth compared to control cells .

How should researchers interpret discrepancies between NCAPD2 mRNA and protein expression data?

Discrepancies between mRNA and protein expression are common in molecular biology research and require careful interpretation when studying NCAPD2:

• Post-transcriptional Regulation: NCAPD2 may be subject to microRNA regulation or RNA methylation. Analysis of the correlation between NCAPD2 expression and RNA methylation regulators has been reported in several tumors .

• Protein Stability: The half-life of NCAPD2 protein may vary depending on cellular context or cancer type. Western blot time-course experiments following transcription or translation inhibition can help determine protein stability.

• Antibody Specificity: Ensure that discrepancies aren't due to antibody cross-reactivity with other condensin complex members. Validation with multiple antibodies targeting different epitopes is recommended.

• Subcellular Localization: NCAPD2 may have different subcellular distribution patterns depending on cell cycle phase or pathological conditions. Immunofluorescence microscopy can help determine if apparent expression differences reflect localization changes rather than absolute protein levels.

• Sample Processing: Differences in tissue preservation methods can affect epitope availability. Standardized protocols for sample collection and processing are essential for consistent results.

What statistical approaches are most appropriate for analyzing NCAPD2 expression in relation to patient outcomes?

The relationship between NCAPD2 expression and patient outcomes requires robust statistical methodology:

How can researchers differentiate between NCAPD2's role in normal mitosis versus cancer-specific functions?

Distinguishing between NCAPD2's normal physiological functions and cancer-specific roles presents a significant challenge requiring sophisticated experimental approaches:

• Comparative Expression Analysis: Compare NCAPD2 expression levels between normal proliferating cells (e.g., lymphocytes, bone marrow cells) and cancer cells with similar proliferation rates to identify cancer-specific overexpression independent of proliferation status.

• Mutation and Variant Analysis: Investigate whether cancer tissues harbor specific NCAPD2 mutations or splice variants that might confer oncogenic properties, using next-generation sequencing approaches.

• Interaction Partner Profiling:

  • Perform immunoprecipitation with NCAPD2 antibodies followed by mass spectrometry to identify differential interaction partners in normal versus cancer cells

  • ChIP-seq analyses to compare genomic binding sites in normal versus transformed cells

• Functional Rescue Experiments:

  • In NCAPD2-depleted cancer cells, compare the effects of reintroducing wild-type NCAPD2 versus mutant forms lacking specific domains

  • This approach can help identify which NCAPD2 functions are essential for cancer progression

• Cell Cycle-Synchronized Experiments:

  • Analyze NCAPD2 function in synchronized cell populations to separate effects on normal mitosis from cancer-specific functions

  • Time-lapse microscopy with fluorescently tagged histones can help visualize chromosome condensation defects

Gene set enrichment analysis (GSEA) has revealed that NCAPD2 is involved in multiple cancer-related pathways beyond its normal mitotic functions, providing insight into its cancer-specific roles .

What emerging technologies can enhance NCAPD2 antibody-based research?

Several cutting-edge technologies can significantly advance NCAPD2 research:

• Proximity Labeling Techniques: BioID or APEX2 fusion with NCAPD2 can identify proteins in close proximity, revealing context-specific interaction partners during different cell cycle phases or in disease states.

• Super-Resolution Microscopy: Techniques like STORM, PALM, or STED combined with NCAPD2 antibodies can provide nanoscale resolution of NCAPD2 localization on chromosomes during condensation.

• Single-Cell Analysis: Single-cell RNA-seq and proteomics can reveal cell-to-cell variation in NCAPD2 expression and function within heterogeneous tumor populations.

• CRISPR-Cas9 Genome Editing: Creating precise mutations in NCAPD2 domains can help dissect structure-function relationships more precisely than traditional knockdown approaches.

• Spatial Transcriptomics: Combining NCAPD2 antibody staining with spatial transcriptomics can reveal how NCAPD2 distribution correlates with gene expression patterns in tumor tissues.

These technologies could help resolve outstanding questions about NCAPD2's specific mechanistic roles in cancer progression and potential as a therapeutic target.

What is the potential of NCAPD2 as a therapeutic target and how can antibodies facilitate drug development?

The consistent overexpression of NCAPD2 across multiple cancer types suggests its potential as a therapeutic target. NCAPD2 antibodies can facilitate drug development through:

• Target Validation: Confirming NCAPD2's role in maintaining cancer cell viability through antibody-based detection following genetic or pharmacological inhibition.

• High-Throughput Screening: Developing cell-based assays with NCAPD2 antibodies to screen for compounds that modulate NCAPD2 expression, localization, or function.

• Mechanism of Action Studies: Using NCAPD2 antibodies to characterize how candidate drugs affect NCAPD2 and the condensin complex.

• Patient Selection Biomarker: NCAPD2 antibodies could help identify patients most likely to respond to treatments targeting chromosome condensation pathways.

• Antibody-Drug Conjugates: Exploring the potential of NCAPD2 antibodies themselves as therapeutics, particularly when coupled with cytotoxic payloads for cancer-targeted delivery.

Drug sensitivity analysis has already identified compounds that show potential activity against NCAPD2-expressing tumors, offering promising avenues for therapeutic development .