CDCA2 Antibody

What is CDCA2 and what are its primary cellular functions?

CDCA2 (Cell division cycle associated 2), also known as Repo-Man, is a cell-cycle protein that recruits protein phosphatase 1 (PP1) to mitotic chromatin at anaphase onset, which is essential for cell proliferation. The CDCA2/PP1 complex is required for chromatin binding and dephosphorylation of histone H3 at Thr3, Ser10, and Ser28. Additionally, this complex is involved in nuclear envelope reformation during mitotic exit for proper progression through the M/G1 transition. CDCA2 may also play a significant role in DNA repair, as the release of CDCA2 from chromatin at sites of DNA damage promotes the activation of DNA damage response pathways .

Why is CDCA2 of interest in cancer research?

CDCA2 has emerged as a protein of significant interest in oncology research because it is highly expressed in multiple cancer types and is considered to be closely related to patient prognosis. Studies have demonstrated upregulated CDCA2 expression in hepatocellular carcinoma (HCC), clear cell renal cell carcinoma (ccRCC), melanoma, neuroblastoma tumors, squamous cell carcinoma, and synovial sarcomas compared to normal tissues . This overexpression pattern suggests CDCA2 may serve as a potential prognostic biomarker and therapeutic target across various cancer types.

What types of CDCA2 antibodies are commonly used in research?

Researchers typically use polyclonal and monoclonal antibodies against CDCA2 in their studies. Polyclonal antibodies, such as the Thermo Fisher Scientific PA5-101297, can detect endogenous levels of total CDCA2 protein. These antibodies are generated by immunizing animals with synthetic peptides or recombinant proteins corresponding to specific regions of human CDCA2. They are designed to recognize epitopes across the protein and are applicable for various experimental techniques including western blotting, immunohistochemistry, and immunofluorescence .

How should CDCA2 antibodies be validated before experimental use?

Proper validation of CDCA2 antibodies is crucial for ensuring experimental reliability. A comprehensive validation approach should include:

• Western blot analysis using positive controls (cell lines known to express CDCA2, such as HepG2 or 786-O) and negative controls (knockout or knockdown cells)

• Peptide competition assays to confirm antibody specificity

• Cross-validation using multiple antibodies targeting different epitopes of CDCA2

• Immunoprecipitation followed by mass spectrometry to confirm the identity of the precipitated protein

• Comparing antibody performance against validated references in the literature

For applications in tissues, validation should also include comparison of staining patterns with known expression profiles from transcriptomic databases .

What are the optimal conditions for using CDCA2 antibodies in western blotting?

For optimal western blot detection of CDCA2, researchers should consider the following protocol parameters:

• Protein extraction: Use radioimmunoprecipitation assay (RIPA) buffer supplemented with protease inhibitor cocktail and phenylmethylsulfonyl protease inhibitor

• Protein quantification: BCA protein assay kit is recommended

• Protein loading: 20 μg protein/lane is typically sufficient

• Gel separation: 7.5–12.5% SDS-PAGE gels work well for CDCA2 detection

• Membrane transfer: Use methanol-activated polyvinylidene membrane

• Blocking: 5% non-fat milk in Tris-buffered saline (pH 7.4) containing 0.1% Tween, for 1 hour at room temperature

• Primary antibody incubation: Anti-CDCA2 antibody (e.g., cat. no. 14976) at 1:1000 dilution overnight at 4°C

• Secondary antibody and detection: HRP-conjugated secondary antibodies followed by enhanced chemiluminescence detection

What are the recommended protocols for immunohistochemical detection of CDCA2?

For immunohistochemical analysis of CDCA2 in tissue sections, the following protocol has been shown to yield reliable results:

• Tissue preparation: Fix tissues in 10% formalin and embed in paraffin

• Sectioning: Prepare 4-5 μm thick sections

• Antigen retrieval: Heat-induced epitope retrieval in citrate buffer (pH 6.0)

• Blocking: Block endogenous peroxidase activity and non-specific binding

• Primary antibody incubation: Anti-CDCA2 antibody (1:100 dilution; e.g., cat. no. 14976) overnight at 4°C

• Secondary antibody incubation: Horseradish peroxidase-labeled secondary antibodies for 15 minutes at room temperature

• Visualization: 3,3′-diaminobenzidine (DAB) for chromogenic development

• Counterstaining: Brief hematoxylin staining (approximately 15 seconds) at room temperature

• Quantification: Analyze positive staining by measuring gray pixels using image analysis software such as Image-Pro Plus

How can CDCA2 expression be effectively silenced for functional studies?

To investigate the functional roles of CDCA2, RNA interference techniques have proven effective. Small interfering RNA (siRNA) duplexes targeting human CDCA2 can be designed and utilized with the following approach:

• siRNA design: Target specific sequences in the CDCA2 mRNA. Examples of effective sequences include:

  • siRNA-1: Forward 5′-CACCUGCCUUUCUAAAUAUTT-3′ and reverse 5′-AUAUUUAGAAAGGCAGGUGTT-3′

  • siRNA-2: Forward 5′-GGGCAAAGGAUCAAGUGAUTT-3′ and reverse 5′-AUCACUUGAUCCUUUGCCCTT-3′

• Negative control: Use non-specific siRNA sequences such as:

  • Forward 5′-UUCUCCGAACGUGUCACGUTT-3′ and reverse 5′-ACGUGACACGUUCGGAGAATT-3′

• Transfection: Use Lipofectamine 2000 or similar reagents following the manufacturer's protocol

• Validation of knockdown: Confirm CDCA2 silencing at both mRNA and protein levels using RT-qPCR and western blotting, respectively. Successful knockdown should achieve at least 60% reduction in expression levels.

• Functional assays: Proceed with cell viability, proliferation, migration, invasion, or apoptosis assays 48-72 hours post-transfection .

What techniques can be used to study CDCA2's role in cell cycle regulation?

To investigate CDCA2's functions in cell cycle regulation, researchers can employ a combination of techniques:

• Synchronization experiments: Block cells at specific cell cycle phases using agents like thymidine (G1/S), nocodazole (M), or serum starvation (G0/G1), then release and analyze CDCA2 expression and localization at different time points

• Live-cell imaging: Use fluorescently tagged CDCA2 to monitor its dynamic localization throughout the cell cycle

• Chromatin immunoprecipitation (ChIP): Identify CDCA2 interactions with chromatin and associated proteins during different cell cycle phases

• Flow cytometry: Analyze cell cycle distribution following CDCA2 knockdown or overexpression using propidium iodide or DAPI staining

• Immunofluorescence co-localization: Examine CDCA2 co-localization with other cell cycle markers or phosphatases at specific phases

• Phosphorylation analysis: Study how cell cycle-dependent phosphorylation (e.g., by Aurora B kinase) affects CDCA2 function and localization .

How can researchers investigate the relationship between CDCA2 and signaling pathways in cancer?

To explore CDCA2's involvement in cancer-related signaling pathways, researchers should consider these methodological approaches:

• Pathway analysis after CDCA2 manipulation:

  • Analyze key signaling proteins after CDCA2 knockdown or overexpression

  • The AKT-mTOR pathway has been specifically implicated in CDCA2-mediated cancer progression

  • Examine the phosphorylation status of AKT (p-AKT), mTOR (p-mTOR), and expression of downstream targets like Cyclin D1

• Rescue experiments:

  • Use specific pathway inhibitors (e.g., AKT inhibitors, mTOR inhibitors) in combination with CDCA2 overexpression

  • Alternatively, activate these pathways in CDCA2 knockdown cells to determine if the phenotype can be rescued

• Co-immunoprecipitation:

  • Identify direct protein-protein interactions between CDCA2 and components of signaling pathways

  • Analyze how these interactions change under various cellular conditions

• Gene expression profiling:

  • Perform RNA-seq or microarray analysis after CDCA2 modulation to identify affected pathways

  • Validate key findings with RT-qPCR and western blotting

How should researchers interpret contradictory CDCA2 expression data across different cancer studies?

When encountering contradictory CDCA2 expression data across cancer studies, researchers should consider several factors for proper interpretation:

• Tissue-specific expression patterns: CDCA2 may have different baseline expression levels and functions in different tissues, leading to apparent contradictions when comparing across cancer types

• Technical variations: Different antibodies, detection methods, and quantification approaches can yield varying results. Always consider the specific antibody clone, dilution, and detection system used

• Sample preparation differences: Variations in fixation methods, antigen retrieval protocols, and tissue processing can affect antibody binding and signal detection

• Heterogeneity within cancer types: Subtype classification, tumor grade, and stage may influence CDCA2 expression patterns

• Contextual evaluation: Integrate CDCA2 expression data with clinical parameters, genetic alterations, and other molecular markers to understand the biological significance

• Statistical rigor: Assess the statistical methods, sample sizes, and validation approaches used in different studies

What are common pitfalls in CDCA2 antibody-based experiments and how can they be avoided?

Researchers should be aware of these common pitfalls and corresponding solutions when working with CDCA2 antibodies:

• Non-specific binding:

  • Problem: Background signals or bands at unexpected molecular weights

  • Solution: Optimize antibody concentration, increase blocking time, use alternative blocking reagents, and include appropriate controls

• Epitope masking:

  • Problem: Reduced or absent signal due to protein modifications or complex formation

  • Solution: Try multiple antibodies targeting different epitopes, optimize antigen retrieval methods

• Cross-reactivity:

  • Problem: Antibody binding to proteins other than CDCA2

  • Solution: Validate specificity using CDCA2 knockdown or knockout samples, perform peptide competition assays

• Quantification challenges:

  • Problem: Difficulty in accurately quantifying expression levels

  • Solution: Use multiple technical replicates, appropriate normalization controls, and standardized image analysis methods

• Batch effects:

  • Problem: Variations between experimental runs

  • Solution: Include standard samples across batches, process experimental and control samples simultaneously

How can researchers validate the specificity of CDCA2 antibody-mediated effects in functional studies?

To ensure that observed phenotypes are specifically due to CDCA2 modulation rather than off-target effects, researchers should implement these validation strategies:

• Multiple siRNA sequences: Use at least two different siRNA sequences targeting distinct regions of CDCA2 mRNA to confirm consistent phenotypes

• Rescue experiments: Restore CDCA2 expression using siRNA-resistant constructs to reverse the knockdown phenotype

• CRISPR/Cas9-mediated knockout: Generate complete CDCA2 knockout cell lines as complementary models to siRNA-mediated knockdown

• Dose-dependent effects: Demonstrate correlation between the degree of CDCA2 knockdown or overexpression and the observed phenotypic changes

• Pathway validation: Confirm that known downstream targets of CDCA2 are affected in a manner consistent with the proposed mechanism

• Alternative methods: Use alternative approaches to modulate CDCA2 function, such as inhibitory peptides or small molecules that disrupt specific protein-protein interactions

How can CDCA2 antibodies be used in patient sample analysis for prognostic purposes?

CDCA2 antibodies can be valuable tools for analyzing patient samples and determining prognostic significance through these methodological approaches:

• Tissue microarray (TMA) analysis:

  • Construct TMAs containing multiple patient samples

  • Perform standardized immunohistochemistry using validated CDCA2 antibodies

  • Implement quantitative scoring systems (H-score, intensity × percentage of positive cells)

  • Correlate CDCA2 expression with clinical parameters and survival outcomes

• Multiplex immunofluorescence:

  • Co-stain for CDCA2 and other cancer markers or cellular compartments

  • Analyze spatial relationships and co-expression patterns

  • Quantify using digital pathology platforms

• Circulating tumor cell analysis:

  • Detect CDCA2 expression in circulating tumor cells

  • Correlate with disease progression and treatment response

• Scoring system standardization:

  • Define clear positive/negative thresholds based on receiver operating characteristic (ROC) curve analysis

  • Consider both staining intensity and percentage of positive cells

  • Ensure blinded evaluation by multiple pathologists

What considerations are important when designing studies to investigate CDCA2 as a therapeutic target?

When exploring CDCA2 as a potential therapeutic target, researchers should consider these important experimental design elements:

• Target validation approaches:

  • Confirm overexpression in patient samples compared to normal tissues

  • Demonstrate cancer cell addiction to CDCA2 through rescue experiments

  • Identify synthetic lethal interactions with common cancer mutations

• Mechanism of action studies:

  • Elucidate the precise mechanism by which CDCA2 inhibition affects cancer cells

  • Determine whether CDCA2 functions primarily through PP1 recruitment or has PP1-independent functions

  • Investigate effects on mitotic progression, DNA damage response, and cell death pathways

• Combination therapy investigation:

  • Test CDCA2 inhibition in combination with standard chemotherapeutics

  • Evaluate synergy with other targeted therapies, particularly those affecting AKT-mTOR signaling

  • Determine sequence-dependent effects (concurrent vs. sequential treatment)

• Resistance mechanisms:

  • Identify potential compensatory pathways that might emerge after CDCA2 inhibition

  • Develop models of acquired resistance

  • Explore rational combination strategies to prevent or overcome resistance

How might single-cell analysis techniques enhance our understanding of CDCA2 function?

Single-cell analysis techniques offer unique advantages for studying CDCA2 function that conventional bulk methods cannot provide:

• Single-cell RNA sequencing (scRNA-seq):

  • Reveal cell-type specific expression patterns of CDCA2

  • Identify rare cell populations with unique CDCA2 expression profiles

  • Map CDCA2 expression to specific cell cycle phases at single-cell resolution

  • Discover co-expression patterns with other genes in individual cells

• Single-cell proteomics:

  • Quantify CDCA2 protein levels in individual cells

  • Correlate with cell cycle markers and signaling pathway components

  • Detect post-translational modifications at the single-cell level

• Single-cell ATAC-seq:

  • Link CDCA2 expression with chromatin accessibility changes

  • Identify potential regulatory elements controlling CDCA2 expression

• Spatial transcriptomics:

  • Map CDCA2 expression within the tumor microenvironment

  • Correlate spatial expression patterns with specific niches or microenvironmental features

• Live-cell single-molecule imaging:

  • Track CDCA2 dynamics in real-time in individual cells

  • Quantify protein-protein interaction kinetics

What are the challenges in developing inhibitors specifically targeting CDCA2?

Developing specific inhibitors against CDCA2 presents several challenges that researchers must address:

• Protein-protein interaction (PPI) targeting:

  • CDCA2 functions primarily through PPIs (especially with PP1), which are traditionally difficult to target with small molecules

  • Requires identification of druggable pockets at interaction interfaces

  • May necessitate alternative approaches like peptide mimetics or proteolysis-targeting chimeras (PROTACs)

• Selectivity concerns:

  • Ensuring specificity for CDCA2 without affecting related proteins

  • Differentiating between CDCA2's interaction with PP1 versus other PP1-interacting proteins

  • Avoiding off-target effects on essential cellular processes

• Context-dependent functions:

  • CDCA2 may have different roles in different cellular contexts

  • Inhibition might need to be tissue or context-specific

  • Understanding which function of CDCA2 (mitotic regulation, DNA damage response, etc.) is most relevant for cancer therapy

• Biomarkers for patient selection:

  • Identifying which patients would benefit most from CDCA2-targeted therapy

  • Developing companion diagnostics to measure CDCA2 dependency

  • Determining appropriate antibodies or other detection methods for patient stratification

What are the key considerations when selecting CDCA2 antibodies for specific research applications?

When selecting CDCA2 antibodies for specific research applications, researchers should consider these critical factors:

• Application compatibility:

  • Verify the antibody has been validated for your specific application (western blot, IHC, IF, flow cytometry, etc.)

  • Review published literature using the antibody in similar applications

• Epitope characteristics:

  • Select antibodies targeting epitopes relevant to your research question

  • Consider whether the epitope might be masked by protein interactions or post-translational modifications

  • For functional studies, choose antibodies targeting domains involved in protein-protein interactions

• Species reactivity:

  • Ensure compatibility with your experimental model (human, mouse, etc.)

  • Verify cross-reactivity if working with multiple species

• Clonality considerations:

  • Monoclonal antibodies offer high specificity for a single epitope but may be sensitive to epitope modifications

  • Polyclonal antibodies recognize multiple epitopes, providing robust detection but potentially more background

• Validation status:

  • Review validation data including western blots, IHC images, and knockout controls

  • Check for independent validation studies beyond manufacturer data

  • Consider antibodies recommended in published CDCA2 studies with rigorous validation

What future directions should CDCA2 antibody research explore?

The field of CDCA2 antibody research presents several promising future directions:

• Development of phospho-specific antibodies:

  • Create antibodies specifically recognizing phosphorylated forms of CDCA2 (e.g., Ser893 phosphorylated by Aurora B)

  • Enable studies of CDCA2 regulation in different cell cycle phases and conditions

• Conformation-specific antibodies:

  • Develop antibodies that distinguish between active and inactive CDCA2 conformations

  • Allow real-time monitoring of CDCA2 functional status

• Super-resolution microscopy compatible antibodies:

  • Optimize antibodies for emerging super-resolution techniques

  • Enable detailed studies of CDCA2 spatial organization and dynamics

• Therapeutic antibody development:

  • Explore the potential for antibody-based targeting of CDCA2 in cancer therapy

  • Investigate antibody-drug conjugates targeting CDCA2-expressing cells

• Multi-omics integration:

  • Combine antibody-based detection with other -omics approaches

  • Develop standardized protocols for integrated analysis of CDCA2 at protein, RNA, and functional levels