CDC20-2 Antibody

What is CDC20 and why is it important in cell cycle research?

CDC20 functions as an essential regulator that promotes mitotic exit by activating the anaphase-promoting complex/cyclosome (APC/C) and monitors kinetochore-microtubule attachment through activation of the spindle assembly checkpoint (SAC) . It serves as a critical control point in mitosis, requiring multiple interactions with APC/C and Mitotic Checkpoint Complex (MCC) subunits to perform these functions . The importance of CDC20 in research stems from its fundamental role in cell division regulation and its implication in various cancers, including glioblastoma, where CDC20 exhibits increased expression in glioblastoma stem-like cells (GSCs) compared to normal astrocytes .

What are the validated applications for CDC20-2 antibody?

The CDC20-2 antibody (such as Proteintech's 84531-2-PBS) has been validated for multiple experimental applications including Cytometric bead array and Indirect ELISA, with demonstrated reactivity to human samples . This recombinant monoclonal antibody is provided in PBS (BSA and azide-free) at a concentration of 1 mg/mL, making it ready for conjugation in various experimental setups . The unconjugated format makes it particularly suitable for developing ELISAs, multiplex assays requiring matched pairs, mass cytometry, and multiplex imaging applications, though optimization is necessary for each specific application .

How should CDC20-2 antibody be stored and handled to maintain optimal activity?

For optimal preservation of activity, CDC20-2 antibody should be stored at -80°C according to manufacturer recommendations . The antibody is typically supplied in PBS buffer without preservatives like BSA or sodium azide, making it conjugation-ready but potentially more susceptible to degradation . When working with the antibody, it's advisable to minimize freeze-thaw cycles, aliquot the stock solution upon first thaw, and maintain sterile conditions during handling. These precautions help preserve the antibody's binding capacity and specificity across experiments.

What are the molecular characteristics of the CDC20 protein targeted by the antibody?

The CDC20 protein targeted by the antibody has a calculated molecular weight of approximately 55 kDa . It is encoded by the CDC20 gene (Gene ID: 991, GenBank Accession Number: BC001088), which produces the cell division cycle 20 homolog (referencing S. cerevisiae) . The protein is recognized in the UniProt database with the identifier Q12834 . CDC20 contains several functional motifs that mediate its interactions with other proteins, including a KEN box, D-box, ABBA motif, C-box, and IR (isoleucine-arginine) motif at the C-terminus, all of which are crucial for its role in cell cycle regulation and APC/C activation .

How can CDC20-2 antibody be used to investigate mitotic checkpoint complex (MCC) formation and function?

CDC20-2 antibody can be employed in co-immunoprecipitation assays to isolate and analyze the components of the mitotic checkpoint complex and their interactions. Research has demonstrated that the MCC can inhibit a second CDC20 molecule that has already bound and activated the APC/C . To investigate this, researchers can use CDC20-2 antibody to immunoprecipitate different CDC20 pools from mitotically arrested cells and analyze the associated proteins by immunoblotting .

For example, researchers have purified different CDC20 complexes using specific antibodies against total CDC20 (reflecting both free CDC20 and MCC-bound CDC20) as well as CDC20 associated with BubR1 and APC/C (reflecting MCC and APC/C-MCC complexes) . Comparative analysis of these different pools using phospho-specific antibodies revealed differential phosphorylation states, with MCC-bound CDC20 showing lower T70 phosphorylation levels compared to total CDC20 . This approach allows for detailed investigation of how post-translational modifications regulate MCC assembly and function.

What experimental approaches can be used to study CDC20's role in cancer cell biology using CDC20-2 antibody?

CDC20-2 antibody serves as a valuable tool in multiple experimental approaches to investigate CDC20's role in cancer:

• Expression analysis: Immunoblotting with CDC20-2 antibody can quantify CDC20 protein levels across different cancer cell types, as demonstrated in studies showing elevated CDC20 expression in glioblastoma stem-like cells compared to normal astrocytes .

• Functional studies following genetic manipulation: After CDC20 knockdown via RNAi or overexpression, CDC20-2 antibody can be used to confirm altered protein levels and investigate downstream effects on cancer cell phenotypes. Studies have shown that CDC20 knockdown in glioblastoma stem cells decreases invasiveness in Matrigel invasion assays .

• Protein interaction studies: CDC20-2 antibody can be used in co-immunoprecipitation experiments to identify cancer-specific protein interactions. Research has revealed CDC20-APC regulation of SOX2 protein in glioblastoma stem cells, where CDC20 promotes SOX2 protein stability by preventing its proteasomal degradation .

• Post-translational modification analysis: Combining CDC20-2 antibody with phospho-specific antibodies enables investigation of how CDC20 phosphorylation status affects its function in cancer cells, potentially revealing cancer-specific regulatory mechanisms.

How can researchers troubleshoot CDC20 detection issues in complex experimental systems?

When encountering difficulties detecting CDC20 in complex experimental systems, researchers should consider several troubleshooting approaches:

• Optimize extraction conditions: CDC20 interacts with multiple protein complexes, so extraction buffers should be optimized to preserve these interactions or specifically disrupt them depending on experimental goals. Include appropriate phosphatase inhibitors if studying phosphorylated forms.

• Consider cell cycle synchronization: CDC20 levels fluctuate during the cell cycle, with peak expression during mitosis. Synchronizing cells using nocodazole or other mitotic arrest agents can enhance detection sensitivity .

• Validate antibody specificity: Confirm antibody specificity using positive controls (cells with known CDC20 expression) and negative controls (CDC20 knockdown cells). Research has shown that even in apparent knockout cells, residual CDC20 peptides may be detected by mass spectrometry, highlighting the importance of thorough validation .

• Use multiple detection methods: Combine immunoblotting with other techniques such as immunofluorescence or mass spectrometry to comprehensively analyze CDC20 expression and localization. Studies have shown that CDC20 localizes to kinetochores, which can be visualized by immunofluorescence using CDC20 antibodies .

What are the methodological considerations for studying CDC20's interaction with the APC/C complex?

Studying CDC20's interaction with APC/C requires careful experimental design:

• Purification of active complexes: To isolate functional CDC20-APC/C complexes, researchers can immunoprecipitate APC/C from CDC20-depleted mitotic cells and reconstitute activity by adding recombinant CDC20 . This approach allows for controlled analysis of how different CDC20 mutations affect APC/C activation.

• Ubiquitylation assays: In vitro ubiquitylation assays using fluorescently labeled substrates (such as securin) provide quantitative measurement of APC/C activity . The reaction typically includes E1-ligase, UbcH10 (E2), ubiquitin, ATP, ATP regenerating system, and the fluorescently-labeled substrate incubated at 37°C, with subsequent analysis by SDS-PAGE and fluorescence scanning .

• Mutational analysis: Systematic mutation of CDC20 functional domains reveals their contribution to APC/C binding and activation. Research demonstrates that removal of the C-terminal IR motif or the C box compromises CDC20 binding to APC/C, causing metaphase arrest followed by cell death .

• Phosphorylation-specific analysis: Phospho-specific antibodies against CDC20 (such as T70) and APC/C components (such as APC3 T447) enable investigation of how phosphorylation regulates complex formation and activity . Quantitative immunoblotting with these antibodies reveals how regulatory factors like BubR1 influence CDC20 phosphorylation state.

How can researchers achieve efficient depletion of endogenous CDC20 for functional studies?

Achieving efficient depletion of endogenous CDC20 presents significant challenges for functional studies because:

• RNAi often provides incomplete knockdown: Traditional siRNA approaches typically fail to deplete CDC20 below the critical threshold required for complete functional analysis . To overcome this limitation, researchers have developed RNAi-sensitive cell lines that exhibit penetrant metaphase arrest following a single RNAi treatment .

• Knockout approaches are challenging: Complete CDC20 knockout is often lethal, making stable knockout cell lines difficult to generate. Studies have shown that apparent knockout clones likely retain residual CDC20 protein undetectable by western blot but identifiable by mass spectrometry . These clones typically show prolonged mitosis (30-70 minutes longer than parental cells) and arrest at metaphase until cell death when treated with CDC20 siRNA .

• Rescue experiments for validation: To confirm knockdown specificity, researchers should perform rescue experiments using RNAi-resistant CDC20 constructs. Studies demonstrate that metaphase arrest caused by RNAi in CDC20 knockout cells can be fully rescued by reintroducing RNAi-resistant YFP-CDC20 .

• Alternative approaches: For acute depletion, researchers can combine small molecule inhibitors of APC/C (such as ProTAME) with CDC20 RNAi to achieve more complete functional inhibition . Additionally, degron-based approaches for targeted protein degradation offer potential alternatives to traditional RNAi.

What controls should be included when using CDC20-2 antibody in protein detection assays?

When using CDC20-2 antibody for protein detection, researchers should include several critical controls:

• Positive control: Include samples with known CDC20 expression, such as mitotically arrested cells where CDC20 levels are high .

• Knockdown/knockout control: Include samples where CDC20 has been depleted via RNAi or CRISPR/Cas9, keeping in mind that complete knockout is challenging due to CDC20's essential nature .

• Loading controls: Use appropriate loading controls (e.g., β-actin, GAPDH) to ensure equal protein loading across samples, particularly important when comparing CDC20 levels between different cell types or conditions.

• Cell cycle controls: Since CDC20 expression varies throughout the cell cycle, include cell cycle markers (such as Cyclin B1, phospho-histone H3) to account for cell cycle-dependent changes in CDC20 levels.

• Antibody validation controls: For new lots of antibody, validation should include peptide competition assays or detection of recombinant CDC20 protein of known concentration to confirm specificity and sensitivity .

• Cross-reactivity assessment: If working with non-human samples, assess potential cross-reactivity since the antibody is primarily validated against human CDC20 .

How can researchers distinguish between different functional pools of CDC20 in experimental systems?

Distinguishing between different functional pools of CDC20 (free CDC20, MCC-bound CDC20, and APC/C-bound CDC20) requires sophisticated experimental approaches:

• Sequential immunoprecipitation: Researchers can use sequential immunoprecipitation with antibodies against CDC20 partners (BubR1, APC/C components) to isolate specific complexes. Studies have employed this approach to purify distinct CDC20 pools and analyze their phosphorylation states .

• Size exclusion chromatography: Different CDC20 complexes can be separated based on size using gel filtration, followed by western blot analysis of fractions to identify complex composition.

• Proximity ligation assays: This technique allows visualization of specific protein-protein interactions in situ, enabling researchers to distinguish between CDC20 interactions with different partners in individual cells.

• Functional mutants as tools: Expression of CDC20 mutants defective in specific interactions can help dissect different functional pools. For example, mutations in the KEN box disrupt BubR1 binding, while mutations in Mad2-binding domains specifically impair MCC formation .

• Fluorescence correlation spectroscopy: For live cell analysis, fluorescently tagged CDC20 combined with correlation spectroscopy can reveal the diffusion characteristics of different CDC20 complexes, providing insights into their size and composition.

How is CDC20-2 antibody used to investigate the role of CDC20 in cancer progression and therapeutic targeting?

CDC20-2 antibody plays a crucial role in investigating CDC20's contribution to cancer progression through several methodological approaches:

• Expression profiling across cancer types: Immunohistochemistry and western blotting with CDC20-2 antibody can establish CDC20 expression patterns across cancer types and correlate with clinical outcomes. Research has demonstrated elevated CDC20 levels in glioblastoma stem-like cells compared to normal astrocytes .

• Mechanistic studies in cancer-specific pathways: Studies using CDC20-2 antibody have revealed that CDC20-APC regulates the stability of SOX2, a key transcription factor in glioblastoma stem cells . This was demonstrated by showing that CDC20 knockdown decreases SOX2 protein levels, which can be reversed by co-expression of RNAi-resistant CDC20 .

• Therapeutic target validation: CDC20-2 antibody can measure changes in CDC20 expression or complex formation following treatment with potential therapeutic agents. Research shows that APC inhibitor ProTAME decreases SOX2 protein levels in glioblastoma stem cells within 4 hours of treatment, suggesting potential therapeutic applications .

• Biomarker development: Quantitative analysis of CDC20 expression using CDC20-2 antibody in patient samples can evaluate its potential as a diagnostic or prognostic biomarker for specific cancer types.

What approaches enable investigation of CDC20 post-translational modifications in normal versus disease states?

Investigating CDC20 post-translational modifications requires specialized techniques:

• Phospho-specific antibodies: Antibodies against specific phosphorylation sites (e.g., CDC20 T70) enable comparison of phosphorylation status between normal and disease states . Research has shown that CDC20 dephosphorylation is regulated by PP2A-B56 recruited by BubR1, and mutations affecting this interaction increase CDC20 T70 phosphorylation .

• Mass spectrometry-based approaches: Immunoprecipitation with CDC20-2 antibody followed by mass spectrometry analysis can identify and quantify multiple post-translational modifications simultaneously. This approach has been used to detect CDC20 peptides in apparent knockout cells, confirming residual protein expression .

• Functional validation of modifications: Expression of phosphomimetic or phospho-deficient CDC20 mutants allows functional assessment of specific modifications. Studies have demonstrated that different CDC20 mutations affecting interaction with APC/C or SAC components result in distinct mitotic phenotypes, from metaphase arrest to accelerated mitosis .

• Inhibitor studies: Combining CDC20-2 antibody detection with kinase or phosphatase inhibitors helps identify enzymes regulating CDC20 modifications. Research has shown that BubR1-recruited PP2A-B56 specifically dephosphorylates CDC20 when it is part of the MCC complex .

How can researchers develop assays to screen for compounds that modulate CDC20 activity for therapeutic purposes?

Developing screening assays for CDC20 modulators requires robust and quantifiable readouts:

• In vitro APC/C activity assays: Reconstituted ubiquitylation assays using purified components (APC/C, CDC20, E1, E2, ubiquitin) and fluorescently labeled substrates provide a direct measure of CDC20-APC/C activity . Compounds inhibiting this activity can be identified through high-throughput screening.

• Cell-based phenotypic assays: Cells expressing fluorescent cell cycle markers can reveal CDC20 inhibition through mitotic arrest or delay. Research has established that CDC20 knockout cells spend 30-70 minutes longer in mitosis compared to parental cells , providing a quantifiable phenotype for screening.

• Protein interaction disruption assays: Assays measuring CDC20 interaction with key partners (APC/C, BubR1, MAD2) can identify compounds that specifically disrupt these interactions. Studies show that MCC inhibits active APC/C within 10 minutes in reconstituted systems , providing a rapid readout for screening.

• Target engagement assays: Cellular thermal shift assays (CETSA) using CDC20-2 antibody can confirm direct binding of compounds to CDC20 in cells, distinguishing direct modulators from indirect effects.

• Downstream functional readouts: Monitoring SOX2 levels and activity using reporter systems can identify compounds that disrupt CDC20-APC regulation of this pathway in cancer cells. Research has demonstrated that CDC20 knockdown substantially decreases SOX2-driven luciferase signal in glioblastoma stem cells .

What emerging technologies might enhance CDC20 research beyond current antibody-based approaches?

Several emerging technologies show promise for advancing CDC20 research:

• CRISPR-based endogenous tagging: Precise insertion of fluorescent or affinity tags at the endogenous CDC20 locus enables visualization and purification of CDC20 complexes without overexpression artifacts.

• Single-molecule imaging techniques: These approaches allow direct visualization of CDC20 dynamics and interactions in living cells at unprecedented resolution, revealing the spatiotemporal regulation of CDC20 function during mitosis.

• Proximity labeling approaches: Technologies like BioID or TurboID fused to CDC20 enable identification of transient or context-specific interactors by labeling proteins in close proximity, potentially revealing novel CDC20 functions and regulatory mechanisms.

• Cryo-electron microscopy: High-resolution structural analysis of CDC20 in complex with APC/C and MCC components provides detailed insights into the molecular mechanisms of CDC20 function and regulation.

• Organoid and patient-derived xenograft models: These more physiologically relevant models enable investigation of CDC20 function in complex tissues and tumors, particularly important for cancer research applications.

How might researchers integrate multi-omics approaches with CDC20-2 antibody studies for comprehensive functional analysis?

Integration of multi-omics approaches with CDC20-2 antibody studies offers powerful strategies for comprehensive analysis:

• Immunoprecipitation coupled with proteomics: CDC20-2 antibody immunoprecipitation followed by mass spectrometry analysis can identify the complete interactome of CDC20 under different conditions, revealing condition-specific interactions and modifications.

• ChIP-seq following CDC20 manipulation: Combining CDC20 knockdown or overexpression with ChIP-seq for transcription factors like SOX2 can reveal how CDC20 indirectly regulates gene expression programs. Research has shown that CDC20-APC controls SOX2 protein stability and transcriptional activity in glioblastoma stem cells .

• Integrated phosphoproteomics: Global phosphoproteomic analysis following CDC20 manipulation can reveal downstream signaling networks affected by CDC20 activity. Studies have demonstrated that BubR1 regulates CDC20 phosphorylation status, affecting MCC formation and function .

• Single-cell multi-omics: Combining CDC20 antibody-based detection with single-cell transcriptomics or proteomics can reveal cell-to-cell heterogeneity in CDC20 expression and function, particularly relevant in heterogeneous tumors.

• Systems biology modeling: Data from multiple omics platforms can be integrated into computational models predicting CDC20 function in complex cellular networks, generating testable hypotheses for further experimental validation.