NCAPH Antibody

What is NCAPH and why is it significant in cancer research?

NCAPH (Non-SMC condensin I complex subunit H) is a regulatory subunit of the condensin I complex involved in chromosome condensation during cell division. Research has demonstrated that NCAPH expression is significantly elevated in various cancer tissues compared to normal tissues, including glioma and lung adenocarcinoma (LUAD) . NCAPH has been identified as promoting malignant characteristics in cancer cells, including proliferation, invasion, migration, and DNA damage repair capabilities . The significance of NCAPH in cancer research lies in its correlation with poor prognosis and its potential as a therapeutic target. In glioma, for instance, NCAPH expression correlates with WHO grade, IDH wild-type status, and non-1p/19q codeletion . Its functional role in the PI3K/AKT signaling pathway makes it particularly relevant for understanding cancer progression mechanisms and developing targeted therapies.

What are the standard protocols for using NCAPH antibody in immunohistochemistry?

For immunohistochemistry (IHC) applications with NCAPH antibody, researchers typically follow this methodological approach:

• Tissue preparation: Fix tissue sections with paraformaldehyde and embed in paraffin.

• Antigen retrieval: Treat sections with hydrogen peroxide (e.g., H2O2) to block endogenous peroxidase activity .

• Blocking: Block non-specific binding using goat serum (e.g., SL038, Solarbio) .

• Primary antibody incubation: Incubate sections with anti-NCAPH primary antibody (typically at 1:100 dilution, e.g., 11,515-1-AP, Proteintech) overnight at 4°C .

• Secondary antibody application: Incubate with HRP-conjugated secondary antibody (e.g., 1:500, #31,460, Thermo Fisher Scientific) at 37°C for 1 hour .

• Signal detection: Detect signals using DAB (e.g., DA1010, Solarbio) .

• Counterstaining: Counterstain sections with hematoxylin .

• Visualization: Scan using a microscope (e.g., BX53, Olympus) .

IHC scoring for NCAPH typically ranges on a scale of 0-5, with higher scores indicating stronger staining intensity and greater protein expression .

What controls should be included when validating NCAPH antibody specificity?

When validating NCAPH antibody specificity, researchers should include these essential controls:

• Positive tissue controls: Include tissues known to express NCAPH, such as cancer tissues from glioma or lung adenocarcinoma samples .

• Negative tissue controls: Include normal adjacent tissues with low NCAPH expression .

• Antibody competition assays: Use purified recombinant NCAPH protein (such as NBP1-88346PEP) as a blocking antigen to verify antibody specificity . This involves pre-incubating the primary antibody with excess NCAPH recombinant protein before application to target tissues.

• Isotype controls: Include matched isotype control antibodies to assess non-specific binding.

• Technical negative controls: Omit primary antibody while maintaining all other steps of the protocol.

• Western blotting validation: Confirm antibody specificity by Western blot, ensuring a single band of appropriate molecular weight.

An antibody with demonstrated specificity should show significantly reduced staining in negative controls and competition assays, while maintaining strong signal in positive controls.

How does NCAPH expression correlate with patient prognosis across different cancer types?

NCAPH expression has been extensively studied for its prognostic significance:

What signaling pathways does NCAPH interact with in promoting tumor progression?

NCAPH promotes tumor progression through several key signaling pathways:

• PI3K/AKT Signaling Pathway: In glioma, NCAPH has been shown to facilitate proliferation, invasion, and migration by promoting the PI3K/AKT signaling pathway . This represents a primary mechanism through which NCAPH exerts its oncogenic effects.

• Cell Cycle Regulation: Co-expression network analysis identified 9 top hub genes (CDK1, BUB1, BUB1B, CCNB1, CCNA2, KIF11, TOP2A, CDC45, CDC20) that interact with NCAPH, all involved in cell cycle regulation . These interactions suggest NCAPH plays a crucial role in cell cycle progression in cancer cells.

• DNA Damage Repair: NCAPH correlates with DNA damage repair ability in glioma cells . Experimental evidence shows that modulating NCAPH expression affects cancer cells' capacity to repair DNA damage.

• Immune Microenvironment Modulation: NCAPH expression is associated with immune and stromal scores in tumor microenvironments. Higher NCAPH expression correlates with increased immune infiltration, particularly affecting Th2 cells (R = 0.898, p < 0.001), while negatively correlating with CD8+ T cells and dendritic cells .

Researchers investigating these pathways should design experiments that incorporate pathway-specific inhibitors alongside NCAPH antibody detection to elucidate the precise molecular mechanisms.

How can NCAPH antibody be used to investigate the cell cycle and proliferation in cancer models?

NCAPH antibody can be strategically employed to investigate cell cycle dynamics and proliferation:

• Flow Cytometry Analysis: Combining NCAPH antibody staining with DNA content analysis using flow cytometry can reveal correlations between NCAPH expression levels and specific cell cycle phases. Research has shown that modulating NCAPH expression affects cell cycle distribution in cancer cells .

• Co-immunostaining with Proliferation Markers: Dual immunofluorescence staining with NCAPH antibody and proliferation markers like Ki-67 enables identification of actively proliferating cells expressing NCAPH . This approach can determine whether NCAPH-positive cells represent a highly proliferative subpopulation.

• Knockdown/Overexpression Studies: Using lentiviral transfection to establish loss-of-function (shRNA-NCAPH) and gain-of-function (OE-NCAPH) cell lines, followed by NCAPH antibody validation, allows researchers to assess proliferation using:

  • CCK-8 assay for cell viability assessment

  • Colony-formation assay for long-term proliferative capacity

  • Cell cycle analysis by flow cytometry

Research findings demonstrate that overexpression of NCAPH significantly enhances proliferation (p < 0.01), while knockdown reduces proliferation (p < 0.01) , confirming its direct role in regulating cancer cell growth.

What are the critical parameters for optimizing NCAPH antibody performance in different applications?

Optimizing NCAPH antibody performance requires careful consideration of several parameters:

When troubleshooting NCAPH antibody applications, researchers should consider:

• Testing multiple antibody clones from different sources

• Validating specificity with recombinant NCAPH protein as a blocking agent

• Optimizing fixation based on sample type (FFPE vs. frozen sections)

• Performing careful titration experiments to determine optimal antibody concentration

How can researchers effectively use NCAPH antibody to assess invasion and migration capabilities of cancer cells?

NCAPH antibody can be effectively incorporated into invasion and migration assays:

• Transwell Migration and Invasion Assays: After manipulating NCAPH expression (overexpression or knockdown), researchers can use NCAPH antibody to confirm expression status before conducting:

  • Standard Transwell assays for migration assessment

  • Matrigel-coated Transwell chambers for invasion evaluation

• Wound Healing Assay: Following cell wound healing assays, NCAPH antibody immunofluorescence can help determine whether cells at the migration front show differential NCAPH expression compared to non-migrating cells .

• Correlation Analysis with EMT Markers: Co-staining of NCAPH with epithelial-mesenchymal transition (EMT) markers can reveal associations between NCAPH expression and invasive phenotype.

Research findings demonstrate that NCAPH overexpression significantly enhances both migration and invasion capabilities of glioma cells, while NCAPH knockdown reduces these properties . The healing rate in wound healing assays is markedly faster in NCAPH-overexpressing cells compared to controls, providing a quantifiable metric for migration capacity .

What approaches can be used to study NCAPH's role in the tumor immune microenvironment?

To investigate NCAPH's role in the tumor immune microenvironment, researchers can employ:

• Immune Cell Profiling: Use NCAPH antibody in combination with immune cell markers to analyze correlations between NCAPH expression and immune cell infiltration. Research has shown NCAPH expression correlates with:

  • Positive association with macrophages, neutrophils, and Th2 cells (R = 0.898, p < 0.001 for Th2)

  • Negative association with CD8+ T cells and dendritic cells

  • No significant association with B cells

• ESTIMATE Algorithm Application: Analyze the relationship between NCAPH expression and immune/stromal scores using the ESTIMATE algorithm. Higher NCAPH expression correlates with higher immune and stromal scores but lower tumor purity scores .

• Single-cell RNA Sequencing Integration: Combine NCAPH antibody-based protein detection with single-cell transcriptomics to identify cell populations with differential NCAPH expression within the tumor microenvironment.

• Functional Co-culture Experiments: Use NCAPH antibody to monitor expression in cancer cells co-cultured with immune cells under different conditions to assess functional interactions.

These approaches can help elucidate how NCAPH contributes to immune evasion mechanisms in cancer and potentially identify new immunotherapeutic strategies.

How can NCAPH be targeted therapeutically, and what role does NCAPH antibody play in drug development?

NCAPH represents a promising therapeutic target, with NCAPH antibody playing crucial roles in drug development:

• Target Validation: NCAPH antibody is essential for confirming target expression in preclinical models and patient samples. Research has established NCAPH as a potential therapeutic target in multiple cancer types, including glioma and LUAD .

• Therapeutic Modulation Approaches:

  • Small Molecule Inhibitors: Five chemicals have been identified that can potentially down-regulate NCAPH expression, as documented in the CTD database .

  • miRNA-based Therapeutics: Eight miRNAs have been identified that potentially down-regulate NCAPH expression .

  • Antibody-Drug Conjugates: NCAPH antibody could potentially be developed into ADCs for targeted therapy, though this requires cell-surface expression or internalization.

• Pharmacodynamic Biomarker: NCAPH antibody serves as a critical tool for measuring target engagement and biological response in drug development.

• Patient Stratification: As NCAPH expression correlates with prognosis, NCAPH antibody-based immunohistochemistry could help identify patients most likely to respond to targeted therapies.

For researchers pursuing NCAPH as a therapeutic target, it's recommended to first validate target dependency through genetic approaches (CRISPR, shRNA) before advancing to pharmacological inhibition strategies.

What methodological approaches can researchers use to study the relationship between NCAPH and DNA damage repair?

To investigate NCAPH's role in DNA damage repair, researchers can implement these methodological approaches:

• DNA Damage Induction and Assessment:

  • Induce DNA damage using radiation or genotoxic agents in cell lines with modulated NCAPH expression

  • Use NCAPH antibody alongside DNA damage markers (γH2AX, 53BP1) to assess correlation between NCAPH expression and damage repair kinetics

  • Quantify repair capacity through comet assay or other DNA damage quantification methods

• Co-immunoprecipitation Studies:

  • Use NCAPH antibody for co-immunoprecipitation to identify interactions with DNA repair proteins

  • Analyze how these interactions change following DNA damage

• Chromatin Immunoprecipitation (ChIP):

  • Perform ChIP with NCAPH antibody to identify genomic regions where NCAPH binds

  • Determine if NCAPH is recruited to sites of DNA damage

• CRISPR-Cas9 Gene Editing:

  • Generate NCAPH knockout or knockdown models

  • Assess DNA repair capacity using reporter assays for homologous recombination or non-homologous end joining

Research findings indicate that NCAPH correlates with DNA damage repair ability in glioma cells , suggesting that these approaches could yield valuable insights into how NCAPH influences genomic stability in cancer cells.

How can researchers validate NCAPH antibody specificity and resolve potential cross-reactivity issues?

Validating NCAPH antibody specificity requires a multi-faceted approach:

• Recombinant Protein Blocking: Use purified NCAPH recombinant protein (such as NBP1-88346PEP with the amino acid sequence: LHCQDYRSELLFPSDVQTLSTGEPLELPELGCVEMTDLKAPLQQCAEDRQICPSLAGFQFTQWDSETHNESVSALVDKFKKNDQVFDINAEVDESDCGDFPDGSLGDDFDANDEPDHT) as a blocking agent in competition assays .

• Genetic Validation: Compare antibody staining between:

  • Wild-type cells/tissues

  • NCAPH knockdown/knockout models

  • NCAPH overexpression models

• Multiple Antibody Comparison: Test multiple antibodies targeting different epitopes of NCAPH (e.g., HPA002647 and HPA003008 as used in Human Protein Atlas) .

• Western Blot Analysis: Perform western blots under reducing and non-reducing conditions to confirm specificity for the expected molecular weight.

• Mass Spectrometry Validation: For advanced validation, immunoprecipitate with NCAPH antibody and confirm pulled-down proteins by mass spectrometry.

If cross-reactivity issues are encountered, researchers should:

• Test alternative antibody clones

• Optimize blocking conditions

• Consider using monoclonal antibodies for higher specificity

• Verify by orthogonal methods (e.g., RNA expression correlating with protein detection)

What are the recommended storage and handling conditions for maintaining NCAPH antibody efficacy?

To maintain NCAPH antibody efficacy, researchers should follow these handling and storage recommendations:

• Storage Temperature: Store antibodies at -20°C for long-term storage. Avoid repeated freeze-thaw cycles that can degrade antibody quality .

• Formulation Considerations: For recombinant NCAPH proteins used as controls or blocking agents, typical formulations include PBS with 1M Urea, pH 7.4, without preservatives .

• Aliquoting Strategy: Upon receipt, aliquot antibodies into single-use volumes to minimize freeze-thaw cycles.

• Working Dilution Preparation:

  • Prepare working dilutions immediately before use

  • For IHC applications, a 1:100 dilution has been validated

  • For other applications, optimize concentration based on specific experimental conditions

• Shipping Conditions: When receiving antibodies, ensure immediate proper storage. NCAPH-related products are typically shipped with polar packs and should be stored immediately at recommended temperatures .

• Stability Assessment: If antibody performance decreases over time, verify activity using positive control samples before troubleshooting experimental procedures.