CDC20-1 Antibody

What is CDC20 and what cellular functions does it regulate?

CDC20 (Cell Division Cycle 20) is an essential cell cycle regulator that serves two primary functions: promoting mitotic exit by activating the Anaphase-Promoting Complex/Cyclosome (APC/C) and monitoring kinetochore-microtubule attachment through activating the Spindle Assembly Checkpoint (SAC). CDC20 contains multiple functional domains, including critical regions like the CRY box that plays a specific role in SAC activation but not APC/C activation . The protein functions as a substrate recognition component of the APC/C ubiquitin ligase complex, directing it to target specific proteins for proteasomal degradation during cell cycle progression.

What are the key physical and molecular properties of CDC20 antibodies?

CDC20 antibodies such as 10252-1-AP are typically generated against CDC20 fusion proteins to ensure specificity. The CDC20 protein has a calculated and observed molecular weight of 55 kDa . Commercial antibodies like 10252-1-AP are often polyclonal rabbit IgG antibodies purified by antigen affinity methods. These antibodies are typically supplied in liquid form with PBS buffer containing 0.02% sodium azide and 50% glycerol at pH 7.3 . For optimal performance, CDC20 antibodies should be stored at -20°C, where they remain stable for approximately one year after shipment.

What are the recommended applications and dilutions for CDC20 antibodies?

CDC20 antibodies can be utilized in multiple experimental applications with specific recommended dilutions:

How should researchers design CDC20 knockdown experiments in cancer cell lines?

For effective CDC20 knockdown studies, researchers should consider the following methodological approach:

• siRNA design and selection: Use validated siRNA sequences targeting CDC20, such as:

  • CDC20 si1: ACCAACCCAUCACCUCAGUtt / ACUGAGGUGAUGGGUUGGUtt

  • CDC20 si2: GGAGCUCAUCUCAGGCCAUtt / AUGGCCUGAGAUGAGCUCCtt

  • CDC20 si3: CAAGAAGGAACAUCAGAAAtt / UUUCUGAUGUUCCUUCUUGtt

• Transfection protocol: Transfect cells using LipofectamineTM RNAiMAX according to manufacturer's protocol, including appropriate negative controls .

• Cell synchronization: For mitosis-specific studies, synchronize cells using double thymidine block before siRNA transfection .

• Validation of knockdown: Confirm CDC20 depletion by Western blot analysis 48-72 hours post-transfection using validated CDC20 antibodies at 1:1000 dilution .

• Functional assays: Follow knockdown with appropriate downstream assays based on research questions (proliferation, migration, cell cycle analysis).
  This approach enables precise analysis of CDC20 function while minimizing off-target effects and maintaining experimental consistency.

What methods are most effective for analyzing CDC20's role in cancer cell proliferation and migration?

A comprehensive analysis of CDC20's role in cancer cell behavior should include multiple complementary techniques:

• Cell proliferation assay: Employ Cell Counting Kit-8 (CCK-8) assays by seeding approximately 1×10⁴ cells per well in 96-well plates followed by CDC20 siRNA transfection. Measure optical density at 450 nm at specific time points (0, 24, 48, and 72 hours) after adding CCK-8 medium and incubating for 4 hours .

• Migration assessment: Conduct wound healing assays by growing cells to 100% confluence in 6-well plates, creating scratches with 10-μL pipette tips after CDC20 knockdown, washing with PBS, and culturing in serum-free medium. Document and quantify wound closure at 0, 24, and 48 hours using ImageJ software .

• Cell cycle analysis: Perform flow cytometry by fixing transfected cells in 75% ethanol overnight at 4°C, staining with PI/RNase buffer for 30 minutes in darkness, and analyzing cell cycle distribution .

• Protein expression analysis: Evaluate downstream effectors through Western blot analysis of key targets including securin, cyclin B1, and cyclin A using specific antibodies (1:5000, 1:3000, and 1:2000 dilutions, respectively) .
  These methodologies collectively provide robust evidence of CDC20's functional impact on cancer cell behavior and underlying mechanisms.

How does the CRY box domain of CDC20 contribute to mitotic checkpoint function?

The CRY box domain of CDC20 plays a critical and specific role in Spindle Assembly Checkpoint (SAC) activation but not in APC/C activation, representing a functional specialization within CDC20 structure. Functional analysis has revealed:

• Structural integrity: Mutations designed to unfold the CDC20 CRY structure (CRY/3A or IPS/3A) result in strong SAC defects, demonstrating the importance of proper folding for checkpoint function .

• Key residues: Among single alanine mutations, R166A alone severely impairs checkpoint function, while single aspartic acid mutations R166D, Y167D, and I168D all produce strong SAC defects, supporting a model of hydrophobic interaction between the CRY box and surrounding residues .

• Regulatory phosphorylation: Ser170 within the CRY box undergoes Plk1-mediated phosphorylation, which facilitates CDC20 degradation during G1 phase, suggesting a regulatory mechanism for temporal control of CDC20 activity .

• Interaction with checkpoint components: The CRY box facilitates proper assembly of the Mitotic Checkpoint Complex (MCC), as mutations R162E/K163E or E180R/D203R in CDC20 significantly impair checkpoint function by disrupting interactions with other checkpoint proteins .
  This evidence establishes the CRY box as a specialized domain enabling CDC20 to participate in mitotic checkpoint signaling through specific protein-protein interactions.

How can researchers experimentally distinguish between CDC20's roles in SAC versus APC/C activation?

To experimentally differentiate between CDC20's dual functions in SAC and APC/C activation, researchers should employ the following methodological approach:

• Domain-specific mutagenesis: Generate a series of CDC20 mutants targeting specific domains:

  • CRY box mutations (R166A, Y167D, I168D) that primarily affect SAC but not APC/C function

  • APC/C binding motif mutations that specifically disrupt CDC20-APC/C interaction

• CRISPR/Cas9 knockout with rescue: Create CDC20 knockout cell lines using CRISPR/Cas9, then complement with RNAi-resistant CDC20 variants to evaluate each mutant's ability to rescue specific functions .

• Mitotic timing analysis: Measure metaphase duration through live-cell imaging of cells expressing fluorescent histone markers to quantify the impact of different CDC20 mutations on mitotic progression .

• Chromosome segregation assessment: Analyze mitotic errors resulting from various CDC20 mutations to determine if defects arise from compromised SAC function versus impaired APC/C activation .

• Biochemical interaction studies: Perform co-immunoprecipitation experiments to assess each mutant's ability to interact with SAC components versus APC/C subunits .
  This multifaceted approach allows researchers to parse the distinct contributions of CDC20 domains to its dual functions in mitotic regulation.

How does CDC20 expression correlate with cancer phenotypes and therapeutic responses?

CDC20 expression has emerged as a critical factor in cancer biology with significant implications for therapeutic strategies:

• Diagnostic biomarker potential: Analysis reveals CDC20 overexpression in multiple cancer types, including Wilms Tumor, where it serves as a candidate diagnostic biomarker distinguishing malignant from normal tissue .

• Correlation with proliferation: High CDC20 expression positively correlates with enhanced cancer cell proliferation, with CDC20 knockdown significantly inhibiting growth in multiple cancer cell lines, suggesting a direct role in promoting malignant growth .

• Impact on therapeutic sensitivity: Aneuploid cancer cells with elevated CDC20 expression show increased sensitivity to Spindle Assembly Checkpoint (SAC) inhibitors, particularly MPS1 inhibitors, presenting CDC20 as a potential predictive biomarker for treatment response .

• Mechanistic basis: High CDC20 levels shorten metaphase duration and increase mitotic errors, resulting in heightened sensitivity to the additional chromosomal instability induced by SAC inhibition therapy .

• Potential therapeutic target: Experimental CDC20 suppression through siRNA significantly impairs cancer cell proliferation and migration by inducing G2/M phase cell cycle arrest, suggesting CDC20 inhibition as a promising therapeutic strategy .
  These findings collectively position CDC20 as both a biomarker for cancer diagnosis and prognosis and a potential therapeutic target or predictive indicator for treatment response.

What techniques are recommended for quantifying CDC20 expression in tumor tissue samples?

For accurate and reproducible quantification of CDC20 expression in tumor tissues, researchers should employ multiple complementary techniques:

• Immunohistochemistry protocol:

  • Prepare 4-μm-thick formalin-fixed, paraffin-embedded tissue sections

  • Perform deparaffinization, rehydration, and antigen retrieval (recommended: TE buffer pH 9.0 or citrate buffer pH 6.0)

  • Block with 3% H₂O₂ and 0.5% BSA

  • Incubate with CDC20 rabbit antibody (1:200 dilution)

  • Apply appropriate detection system and counterstain

  • Quantify using Histochemistry score [H score = ∑(PI × I)] where PI is percentage of cells and I is intensity (1-3)

• Western blot quantification:

  • Extract total protein using RIPA buffer with protease inhibitors

  • Determine concentrations via BCA assay

  • Separate proteins by SDS-PAGE and transfer to PVDF membranes

  • Block with 5% BSA and incubate with CDC20 antibody (1:1000)

  • Detect using chemiluminescence and analyze band intensity with software like Quantity One

• Quantitative PCR:

  • Extract RNA from tissues using standard protocols

  • Synthesize cDNA and perform qPCR with CDC20-specific primers

  • Normalize expression to appropriate housekeeping genes

  • Calculate relative expression using the 2^(-ΔΔCT) method
    These methodologies provide complementary data on CDC20 protein levels and localization, enabling robust assessment of CDC20 status in clinical samples.

What are common technical challenges when working with CDC20 antibodies and how can they be addressed?

When working with CDC20 antibodies, researchers frequently encounter several technical challenges that can be systematically addressed:

• Specificity concerns:

  • Problem: Detection of non-specific bands in Western blot

  • Solution: Validate antibody specificity using CDC20 knockdown or knockout samples as negative controls; optimize antibody concentration (1:2000-1:14000 for WB); include proper blocking (5% BSA) and washing steps

• Variable signal intensity:

  • Problem: Inconsistent signal strength across experiments

  • Solution: Standardize protein loading (1.0-3.0 mg total protein); optimize antibody concentration for each application; ensure consistent transfer conditions; use fresh detection reagents; consider enhanced chemiluminescence for low-abundance samples

• Immunohistochemistry optimization:

  • Problem: Weak or non-specific tissue staining

  • Solution: Test different antigen retrieval methods (TE buffer pH 9.0 and citrate buffer pH 6.0); optimize antibody dilution (1:50-1:500); extend incubation times; use polymer-based detection systems for signal amplification

• Cell-type dependent performance:

  • Problem: Variable results across different cell lines

  • Solution: Validate antibody in each specific cell type; adjust protein extraction methods based on cell type; optimize antibody concentration for each cell line; consider using cell lines with confirmed CDC20 expression (HeLa, Jurkat, HepG2)

• Storage and stability issues:

  • Problem: Diminished antibody performance over time

  • Solution: Store at recommended temperature (-20°C); avoid repeated freeze-thaw cycles; aliquot antibody upon receipt; add 0.1% BSA for smaller volume preparations; use within one year of shipment
    Addressing these challenges through systematic optimization will significantly improve experimental outcomes when working with CDC20 antibodies.

How can CDC20 antibodies be applied in studying the relationship between mitotic duration and chromosomal instability?

CDC20 antibodies can be strategically employed to investigate the complex relationship between mitotic duration and chromosomal instability through the following methodological approaches:

• Combined immunofluorescence and live-cell imaging:

  • Co-stain cells with CDC20 antibodies (1:200-1:800 dilution) and markers for kinetochores, spindle, and chromosomes

  • Perform time-lapse imaging of fluorescently-tagged histones to track chromosome movements

  • Quantify CDC20 levels at kinetochores and correlate with metaphase duration and segregation errors

• CDC20 manipulation and mitotic timing analysis:

  • Deplete CDC20 using siRNA or overexpress CDC20 using expression vectors

  • Measure metaphase duration through live-cell imaging

  • Correlate CDC20 levels with mitotic timing and subsequent chromosomal abnormalities

  • Document that CDC20 depletion prolongs metaphase and reduces mitotic errors, while high CDC20 expression shortens metaphase and increases errors

• CDC20-APC/C interaction studies in response to SAC inhibitors:

  • Use CDC20 antibodies (0.5-4.0 μg) for immunoprecipitation of CDC20-APC/C complexes

  • Analyze complex formation in the presence of MPS1 inhibitors or other SAC inhibitors

  • Correlate complex formation with mitotic progression and chromosomal stability

• Quantitative analysis of CDC20 and downstream targets:

  • Perform Western blot analysis of CDC20 alongside key targets like securin, cyclin B1, and cyclin A

  • Correlate protein levels with cell cycle distribution measured by flow cytometry

  • Track changes in these markers in response to SAC inhibitors to understand mechanism of increased sensitivity in high-CDC20 cells
    These approaches collectively enable researchers to dissect how CDC20 levels influence mitotic timing, chromosomal stability, and sensitivity to SAC inhibitors, providing insights into potential therapeutic strategies targeting mitotic regulation in cancer.

How might CDC20 expression serve as a predictive biomarker for response to mitotic checkpoint inhibitors?

Recent research suggests CDC20 expression has significant potential as a predictive biomarker for response to mitotic checkpoint inhibitors, particularly MPS1 inhibitors:

• Mechanistic basis: High CDC20 expression in aneuploid cancer cells shortens metaphase duration and increases mitotic errors, creating a vulnerability to additional chromosomal instability induced by SAC inhibition therapy. This mechanism suggests CDC20 levels could predict which tumors will be most sensitive to these agents .

• Biomarker validation approach:

  • Quantify CDC20 expression in patient-derived xenografts or tumor samples using standardized IHC protocols with 1:200 antibody dilution

  • Correlate expression levels with in vitro and in vivo response to MPS1 inhibitors

  • Establish threshold CDC20 expression levels predictive of treatment response

  • Validate in prospective clinical studies comparing outcomes between high and low CDC20-expressing tumors

• Combination therapy potential: Experimental evidence suggests CDC20 expression levels might predict which tumors would benefit from combinatorial approaches targeting both CDC20 and the spindle assembly checkpoint, potentially enabling more personalized treatment strategies .

• Clinical implementation: Development of a standardized CDC20 expression assay using validated antibodies could enable patient stratification in clinical trials of mitotic checkpoint inhibitors, potentially improving response rates through targeted patient selection.
  This emerging research direction could significantly impact precision oncology by identifying patients most likely to benefit from mitotic checkpoint inhibitor therapy based on tumor CDC20 expression profiles.

What are the current challenges in developing CDC20-targeted cancer therapies?

The development of CDC20-targeted cancer therapies faces several significant challenges that researchers must address:

• Target specificity concerns:

  • CDC20 plays essential roles in normal cell division, making complete inhibition potentially toxic to normal dividing tissues

  • Developing inhibitors with a therapeutic window that selectively affects cancer cells remains challenging

  • Current approaches using siRNA (sequences targeting ACCAACCCAUCACCUCAGU, GGAGCUCAUCUCAGGCCAU, or CAAGAAGGAACAUCAGAAA) demonstrate proof-of-concept but have limited clinical application

• Mechanism-based resistance:

  • Cancer cells may adapt to CDC20 inhibition through compensatory mechanisms

  • Potential upregulation of CDC20 homologs or alternative APC/C activators

  • Mutations in CDC20 binding domains (like the CRY box) could confer resistance to targeted therapies

• Biomarker development:

  • Need for standardized methods to quantify CDC20 expression in clinical samples

  • Current immunohistochemistry protocols require optimization across different tumor types

  • Threshold determination for "high" versus "low" CDC20 expression as predictive biomarkers

• Combination strategy optimization:

  • Determining optimal combinations of CDC20-targeted therapies with conventional or other targeted approaches

  • Understanding sequence-dependent effects when combining with SAC inhibitors

  • Identifying synergistic versus antagonistic combination strategies

• Clinical trial design:

  • Patient selection strategies based on CDC20 expression or functional status

  • Appropriate endpoints for trials targeting mitotic regulators (response rate may underestimate clinical benefit)

  • Biomarker-driven adaptive trial designs to account for heterogeneous CDC20 biology across cancer types Addressing these challenges requires multidisciplinary approaches combining structural biology, medicinal chemistry, cancer biology, and clinical medicine to develop effective CDC20-targeted therapeutic strategies with acceptable safety profiles.