CDC20-3 Antibody

What is CDC20 and what are its primary cellular functions?

CDC20 functions as an E3 ubiquitin ligase that plays crucial roles in cell cycle progression, particularly during the metaphase-to-anaphase transition. It serves as an essential activator of the anaphase-promoting complex (APC), facilitating the degradation of specific cell cycle proteins via the ubiquitin-proteasome pathway. Beyond cell cycle regulation, CDC20 participates in apoptotic processes and has recently been shown to protect cardiomyocytes from doxorubicin-induced injury by modulating CCDC69 degradation . CDC20 is highly expressed in various malignant tumors, suggesting its involvement in tumor occurrence and progression, and it protects diverse tumor cells from apoptosis .

What applications are CDC20 antibodies validated for in research settings?

CDC20 antibodies have been validated for numerous research applications, with specific antibodies like 10252-1-AP demonstrating effectiveness in Western Blot (WB), Immunohistochemistry (IHC), Immunofluorescence/Immunocytochemistry (IF/ICC), Immunoprecipitation (IP), Co-Immunoprecipitation (CoIP), and ELISA techniques . Published literature confirms their utility in knockdown/knockout validation studies, with at least 9 publications documenting such applications . These antibodies have shown reactivity with human samples, including detection in HEK-293, HeLa, PC-3, HL-60, Jurkat, and HepG2 cells via Western blotting, and in human breast cancer, colon cancer, and urothelial carcinoma tissues via immunohistochemistry .

What is the optimal sample preparation method for CDC20 antibody detection in fixed tissues?

For optimal CDC20 antibody detection in fixed tissues, antigen retrieval plays a critical role. The recommended protocol involves antigen retrieval with TE buffer at pH 9.0, although citrate buffer at pH 6.0 can serve as an alternative . This retrieval step is particularly important when performing immunohistochemistry on formalin-fixed, paraffin-embedded tissues such as human breast cancer, colon cancer, or urothelial carcinoma samples . The antibody dilution should be optimized between 1:50-1:500 for IHC applications, with the specific dilution being sample-dependent to achieve optimal signal-to-background ratios.

How does CDC20 ubiquitinate CCDC69 and what domains are involved in this interaction?

CDC20 directly ubiquitinates CCDC69 in myocardial cells through specific domain interactions. Research has identified that two segments of CDC20—amino acids 168–477 and 168–499—clearly mediate CCDC69 ubiquitination, while the amino acids 1–167 fragment weakly mediates this process . The interaction between CDC20 and CCDC69 has been confirmed through co-immunoprecipitation experiments where CCDC69 was efficiently precipitated by anti-CDC20 antibodies but not by IgG controls . CDC20 overexpression markedly enhances the ubiquitination of CCDC69, whereas CDC20 knockdown substantially suppresses this ubiquitination . This ubiquitination reduces the half-life of CCDC69, a process that can be reversed by the addition of the proteasome inhibitor MG132, indicating a proteasome-dependent degradation mechanism .

What considerations should be made when designing CDC20 knockdown/knockout validation experiments?

When designing CDC20 knockdown/knockout validation experiments, researchers should consider several critical factors. First, validation of knockdown/knockout efficiency should be performed using multiple techniques, including Western blot and qPCR. Based on published studies, CDC20 knockdown using siRNA (Ad-siCDC20) or pharmacological inhibition using apcin has been shown to exacerbate doxorubicin-induced cardiomyocyte apoptosis . For myocardium-specific knockout models, approaches such as the CDC20-myh6 mouse model with tamoxifen-induced gene recombination have been successfully employed . Researchers should allow adequate time (approximately 2 weeks) for tamoxifen clearance before experimental interventions. Additionally, control for potential compensatory mechanisms, as CDC20 plays crucial roles in cell cycle regulation, and its complete knockout may have pleiotropic effects on cellular physiology.

How does CDC20 expression affect doxorubicin efficacy in cancer models versus cardioprotection?

CDC20 expression demonstrates a fascinating dual role in doxorubicin treatment contexts. Cardiomyocyte-specific overexpression of CDC20 significantly protects against doxorubicin-induced cardiac injury by reducing cardiomyocyte apoptosis, inflammation, fibrosis, and cell atrophy . Importantly, this cardioprotective effect occurs without compromising doxorubicin's antitumor efficacy. In tumor models where tumors reached approximately 100 mm³ before doxorubicin administration, both the DOX+tumor and DOX+AAV9-CDC20+tumor groups showed significantly decreased tumor volumes compared to the tumor-only group, with no significant differences between the two doxorubicin-treated groups . Mechanistically, this is evidenced by similar upregulation of pro-apoptotic proteins (BAX), downregulation of proliferation markers (PCNA), and increased caspase 3 activity in both doxorubicin-treated groups . This has been demonstrated in multiple cancer models, including melanoma, suggesting that CDC20's cardioprotective role can be leveraged without diminishing doxorubicin's anticancer properties .

What are the optimal dilution ratios for CDC20 antibodies across different experimental applications?

The optimal dilution ratios for CDC20 antibodies vary significantly depending on the specific application and must be titrated for each experimental system. For Western Blot applications, a dilution range of 1:2000-1:14000 is recommended . Immunohistochemistry applications typically require more concentrated antibody, with recommended dilutions between 1:50-1:500 . For Immunofluorescence/Immunocytochemistry applications, dilutions between 1:200-1:800 have been effective . When performing Immunoprecipitation, using 0.5-4.0 μg of antibody for 1.0-3.0 mg of total protein lysate is suggested . These recommendations serve as starting points, and researchers should perform optimization experiments with positive and negative controls to determine the ideal dilution for their specific samples and experimental conditions.

What controls are essential when using CDC20 antibodies in protein interaction studies?

When conducting protein interaction studies with CDC20 antibodies, multiple controls are essential to ensure result validity. First, include appropriate negative controls such as IgG isotype controls to account for non-specific binding, as demonstrated in co-immunoprecipitation experiments where CCDC69 was not precipitated by IgG controls . Second, incorporate relevant positive controls using cell lines known to express CDC20, such as HEK-293, HeLa, or Jurkat cells . Third, validate antibody specificity through knockdown/knockout approaches, comparing wild-type to CDC20-depleted samples. Fourth, when investigating ubiquitination, include proteasome inhibitors like MG132 as experimental controls to prevent protein degradation, as demonstrated in CDC20-CCDC69 interaction studies . Finally, for co-immunoprecipitation experiments, perform reciprocal pulldowns (e.g., if pulling down with CDC20 antibody, also perform the reverse experiment pulling down with the interaction partner's antibody) as was done with Flag-CDC20 and myc-CCDC69 to confirm their interaction .

How can researchers effectively detect CDC20-mediated ubiquitination of target proteins?

To effectively detect CDC20-mediated ubiquitination of target proteins, researchers should employ a multi-faceted approach. Begin by overexpressing tagged versions of both CDC20 (e.g., Flag-CDC20) and the target protein (e.g., myc-CCDC69) in appropriate cell lines, followed by immunoprecipitation using antibodies against the target protein's tag . Immunoblotting with anti-ubiquitin antibodies can then reveal ubiquitination levels, which should increase with CDC20 overexpression and decrease with CDC20 knockdown . Include proteasome inhibitors (e.g., MG132) in parallel experiments to prevent degradation of ubiquitinated proteins and enhance detection . To identify specific domains involved in ubiquitination, transfect cells with full-length and truncated versions of CDC20, as was done to identify that amino acids 168–477 and 168–499 segments most effectively mediate CCDC69 ubiquitination . Monitor protein half-life using cycloheximide chase assays, comparing degradation rates in the presence and absence of CDC20 overexpression . This comprehensive approach allows for thorough characterization of CDC20-mediated ubiquitination processes.

How can researchers address non-specific binding when using CDC20 antibodies in complex tissue samples?

When encountering non-specific binding of CDC20 antibodies in complex tissue samples, several optimization strategies should be implemented. First, titrate antibody concentration more carefully, as the recommended dilution range (1:50-1:500 for IHC) is quite broad and sample-dependent . Second, optimize blocking conditions by testing different blocking agents (BSA, normal serum, casein) and increasing blocking time. Third, ensure proper antigen retrieval, following the recommended protocol with TE buffer at pH 9.0 or alternatively with citrate buffer at pH 6.0 . Fourth, increase washing steps duration and frequency between antibody incubations. Fifth, pre-absorb the primary antibody with recombinant CDC20 protein to reduce non-specific binding. Sixth, validate antibody specificity using positive control tissues known to express CDC20 (e.g., human breast cancer, colon cancer, or urothelial carcinoma tissues) alongside negative controls such as CDC20 knockout tissues or tissues known not to express CDC20. Finally, consider using alternative detection systems or more specific secondary antibodies to reduce background signals.

What factors might lead to discrepancies in CDC20 detection between different experimental methods?

Discrepancies in CDC20 detection between different experimental methods can arise from several factors. First, different antibody epitopes may be differentially accessible depending on the technique—for instance, epitopes masked in native protein conformation (affecting IP) but exposed after denaturation (affecting WB). Second, post-translational modifications of CDC20, particularly ubiquitination and phosphorylation, can affect antibody recognition in different contexts. Third, protein-protein interactions may mask epitopes in certain contexts; for example, CDC20's interaction with CCDC69 or other binding partners may interfere with antibody binding . Fourth, sample preparation methods vary significantly between techniques—formalin fixation for IHC may alter epitope accessibility compared to gentle lysis for IP. Fifth, CDC20 expression levels vary considerably across cell types and tissues; it is highly expressed in various malignant tumors but may be less abundant in normal tissues . Sixth, detection sensitivity differs between methods, with Western blotting generally being more sensitive than IHC. Finally, subcellular localization of CDC20 can affect detection in imaging-based methods like IF/ICC, potentially leading to apparent discrepancies when compared with biochemical methods.

How should researchers interpret conflicting data regarding CDC20 expression levels in different experimental systems?

When faced with conflicting data regarding CDC20 expression levels across different experimental systems, researchers should consider several interpretive frameworks. First, evaluate cell cycle status differences between experimental systems, as CDC20 expression fluctuates throughout the cell cycle, peaking during mitosis. Second, assess tissue context influences; CDC20 is highly expressed in various malignant tumors but may have different expression patterns in normal tissues . Third, consider regulatory mechanisms affecting CDC20 in different systems—its expression can be influenced by various transcription factors, microRNAs, and post-translational modifications. Fourth, examine technical variables such as antibody specificity, detection methods, and normalization strategies used across studies. Fifth, analyze whether differences in expression are absolute or relative—small relative changes may still be biologically significant. Sixth, investigate whether conflicting data might reflect alternative splicing variants or post-translationally modified forms of CDC20 that are differentially detected by various antibodies. Finally, design validation experiments using multiple techniques (WB, qPCR, IHC) and multiple antibodies targeting different epitopes to resolve discrepancies and establish definitive expression patterns.

How can CDC20 antibodies be applied to study its role in doxorubicin-induced cardiotoxicity?

CDC20 antibodies can be applied to investigate doxorubicin-induced cardiotoxicity through multiple experimental approaches. Immunohistochemistry and immunofluorescence using CDC20 antibodies can visualize changes in CDC20 expression and localization in cardiac tissues following doxorubicin treatment . Western blotting can quantify CDC20 protein levels in cardiomyocytes under normal and doxorubicin-treated conditions, revealing downregulation patterns as observed in transcriptomic analyses . Researchers can employ co-immunoprecipitation with CDC20 antibodies to identify interaction partners in cardiac tissue, particularly focusing on CCDC69 and other potential targets involved in cardioprotection . For mechanistic studies, combining CDC20 antibodies with ubiquitination assays allows for the assessment of CDC20's E3 ligase activity toward specific substrates like CCDC69 in cardiomyocytes . In vivo, immunostaining of cardiac sections from CDC20-myh6 knockout mice or AAV9-cTNT-(si)CDC20-treated mice can validate CDC20's role in protecting against doxorubicin-induced apoptosis, inflammation, fibrosis, and cell atrophy . These applications collectively provide comprehensive insights into CDC20's cardioprotective mechanisms.

What methodological approaches can detect CDC20-CCDC69 interaction in different cellular contexts?

Detecting CDC20-CCDC69 interactions across different cellular contexts requires multiple complementary methodological approaches. Co-immunoprecipitation represents a foundational technique, where CDC20 antibodies can pull down CCDC69 (or vice versa) from cell lysates, as demonstrated in experiments where CCDC69 was efficiently precipitated by anti-CDC20 antibodies but not by IgG controls . For enhanced detection, researchers can use tagged versions of both proteins (e.g., Flag-CDC20 and myc-CCDC69) followed by immunoprecipitation with anti-Flag or anti-Myc antibodies . Proximity ligation assays (PLA) can visualize CDC20-CCDC69 interactions in situ within intact cells or tissues, providing spatial information about where these interactions occur. For studying the dynamic nature of these interactions, fluorescence resonance energy transfer (FRET) or bioluminescence resonance energy transfer (BRET) approaches with fluorescently labeled CDC20 and CCDC69 can be employed. To validate direct binding, in vitro pull-down assays using recombinant purified proteins can confirm direct interaction without cellular cofactors. Finally, to map interaction domains, researchers can use truncated versions of CDC20 (as was done to identify that amino acids 168–477 and 168–499 segments most effectively mediate CCDC69 interaction) combined with these detection methods.

How might CDC20 antibodies contribute to developing cardioprotective strategies for cancer patients receiving doxorubicin?

CDC20 antibodies can significantly contribute to developing cardioprotective strategies for cancer patients receiving doxorubicin through several research pathways. First, they enable monitoring of CDC20 expression levels in patient-derived cardiac samples before and after doxorubicin treatment, potentially identifying patients at higher risk for cardiotoxicity based on CDC20 downregulation patterns . Second, CDC20 antibodies facilitate high-throughput screening of compounds that might stabilize CDC20 expression or enhance its activity in cardiomyocytes without affecting its tumor-related functions . Third, they allow for mechanistic studies to identify the precise signaling pathways through which CDC20 provides cardioprotection, particularly focusing on its ubiquitination targets like CCDC69 . Fourth, CDC20 antibodies can aid in developing predictive biomarker panels for doxorubicin-induced cardiotoxicity by correlating CDC20 levels with cardiotoxicity outcomes. Fifth, they enable validation of cardiomyocyte-specific CDC20 overexpression approaches (such as AAV9-CDC20 delivery) that have shown promise in animal models for protecting against doxorubicin-induced cardiac injury without compromising antitumor efficacy . Finally, CDC20 antibodies can help distinguish between cardiac and tumor-specific CDC20 functions, potentially leading to targeted therapeutic approaches that enhance cardioprotection while maintaining doxorubicin's anticancer properties .

What quantitative methods provide the most reliable assessment of CDC20 protein levels in tissue samples?

Reliable quantitative assessment of CDC20 protein levels in tissue samples requires careful selection and optimization of methodological approaches. Western blotting with appropriate normalization to housekeeping proteins (e.g., GAPDH, β-actin) provides relative quantification of CDC20 protein levels and has been extensively validated across multiple cell types . For absolute quantification, enzyme-linked immunosorbent assay (ELISA) using calibrated standards offers precise measurement of CDC20 concentration, though commercial CDC20 ELISA kits require thorough validation . Mass spectrometry-based approaches, particularly selected reaction monitoring (SRM) or parallel reaction monitoring (PRM), provide highly accurate absolute quantification without antibody dependency, though they require specialized equipment and expertise. Digital spatial profiling combines immunofluorescence with digital counting technologies to quantify CDC20 while preserving spatial context within heterogeneous tissues. For clinical samples, immunohistochemistry with digital image analysis using H-score or Allred scoring systems allows for semi-quantitative assessment of CDC20 expression patterns . When comparing CDC20 levels across different experimental conditions, consistent sample preparation and simultaneous processing are crucial regardless of the quantification method chosen.

How should researchers optimize CDC20 immunoprecipitation protocols for detecting transient protein interactions?

Optimizing CDC20 immunoprecipitation protocols for detecting transient protein interactions requires careful attention to several key factors. First, implement chemical crosslinking with membrane-permeable agents (e.g., DSP, formaldehyde) prior to cell lysis to stabilize transient CDC20 interactions, as demonstrated in CDC20-CCDC69 interaction studies . Second, modify lysis conditions to preserve protein interactions by using gentle, non-denaturing buffers with low detergent concentrations while maintaining sufficient stringency to reduce non-specific binding. Third, supplement lysis buffers with proteasome inhibitors like MG132 to prevent degradation of ubiquitinated interaction partners, as CDC20 functions as an E3 ubiquitin ligase targeting proteins for degradation . Fourth, conduct immunoprecipitation at 4°C with shortened incubation times to capture more transient interactions while minimizing loss of stable complexes. Fifth, optimize antibody concentration through titration experiments, using 0.5-4.0 μg of CDC20 antibody for 1.0-3.0 mg of total protein lysate as a starting point . Sixth, enhance detection sensitivity by employing more sensitive detection methods such as chemiluminescence with longer exposure times or immunoblotting with high-sensitivity fluorescent secondary antibodies. Finally, validate interactions through reciprocal co-immunoprecipitation experiments as was performed with Flag-CDC20 and myc-CCDC69 .