CDC27B Antibody

What is CDC27 and what is its biological significance?

CDC27 (also known as ANAPC3) is a critical component of the anaphase-promoting complex/cyclosome (APC/C), a cell cycle-regulated E3 ubiquitin ligase that controls progression through mitosis and the G1 phase of the cell cycle . The APC/C complex functions by mediating ubiquitination and subsequent degradation of target proteins, primarily through the formation of 'Lys-11'-linked polyubiquitin chains, and to a lesser extent, 'Lys-48'- and 'Lys-63'-linked polyubiquitin chains . Additionally, the APC/C complex catalyzes the assembly of branched 'Lys-11'-/'Lys-48'-linked ubiquitin chains on target proteins, marking them for proteasomal degradation . This activity is essential for proper cell cycle progression, particularly during the metaphase-to-anaphase transition and mitotic exit.

What are the main applications of CDC27 antibodies in research?

CDC27 antibodies are versatile research tools with several key applications:

• Western Blotting (WB): Used to detect and quantify CDC27 protein expression in cell or tissue lysates, typically identifying a band at approximately 90 kDa .

• Immunoprecipitation (IP): Enables isolation of CDC27 protein complexes from cell lysates for further analysis of protein-protein interactions within the APC/C complex .

• Immunohistochemistry (IHC): Allows visualization of CDC27 expression patterns and subcellular localization in tissue sections, which has proven valuable in cancer research .

• Functional Studies: Used in combination with genetic manipulation to investigate CDC27's role in cell cycle regulation, cancer progression, and developmental disorders .

How do I determine the appropriate CDC27 antibody for my specific research application?

Selection of the appropriate CDC27 antibody should be based on several criteria:

• Target Species: Ensure the antibody is validated for your species of interest. For example, antibodies like ab72214 are validated for human samples .

• Application Compatibility: Verify the antibody is suitable for your intended application (WB, IP, IHC). Some antibodies perform well in multiple applications while others are application-specific .

• Epitope Recognition: Consider which domain or region of CDC27 you wish to target. For instance, some antibodies recognize the extreme C-terminus (amino acids 814-823) , while others may target different regions.

• Validation Status: Review published literature citing the antibody and manufacturer validation data to ensure reliable performance in your experimental conditions.

• Polyclonal vs. Monoclonal: Polyclonal antibodies (like ab72214) offer multiple epitope recognition but potential batch variability, while monoclonal antibodies provide greater specificity and reproducibility .

What are the optimal conditions for CDC27 antibody use in Western blotting?

For optimal Western blotting results with CDC27 antibodies:

• Sample Preparation: Lyse cells in a buffer containing 50 mM HEPES (pH 7.4), 150 mM NaCl, 0.5% NP-40, 1 mM EDTA, 1 mM Na₃VO₄, and protease inhibitors (1 mM aprotinin, 1 mM leupeptin, 1 mM PMSF) .

• Protein Loading: Load 20-40 μg of total protein per lane, as CDC27 is typically expressed at moderate levels in most cell types.

• Antibody Dilution: Use a 1:1000 to 1:2000 dilution for primary antibody incubation, though optimal dilution may vary by antibody and should be empirically determined .

• Incubation Conditions: Incubate with primary antibody at 4°C overnight or room temperature for 2 hours for best signal-to-noise ratio.

• Detection Method: Enhanced Chemiluminescence (ECL) Plus systems work effectively for visualizing CDC27, which typically appears as a band of approximately 90 kDa .

• Controls: Include both positive controls (cell lines known to express CDC27) and negative controls (CDC27 knockdown/knockout samples if available) to validate specificity.

What protocol should I follow for immunoprecipitation using CDC27 antibodies?

For effective immunoprecipitation of CDC27:

• Cell Lysis: Lyse cells at 4°C for 30 minutes in buffer containing 50 mM HEPES (pH 7.4), 150 mM NaCl, 0.5% NP-40, 1 mM EDTA, 1 mM Na₃VO₄, and protease inhibitors (1 mM aprotinin, 1 mM leupeptin, 1 mM PMSF) .

• Pre-clearing: Pre-clear lysates with protein G-sepharose for 1 hour at 4°C to reduce non-specific binding.

• Antibody Incubation: Incubate cleared lysates (0.5-2 mg total protein) with CDC27 antibody for 4 hours at 4°C .

• Bead Incubation: Add protein G-sepharose beads and incubate for an additional 2 hours at 4°C with gentle rotation .

• Washing: Wash the immunoprecipitates extensively (4-5 times) with lysis buffer to remove non-specifically bound proteins.

• Elution: Elute bound proteins by boiling in SDS sample buffer for 5 minutes.

• Analysis: Analyze the immunoprecipitated proteins by immunoblotting with appropriate antibodies to detect CDC27 and its interaction partners .

What is the recommended protocol for CDC27 immunohistochemical staining?

For optimal immunohistochemical detection of CDC27:

• Sample Preparation: Use 3-μm tissue sections mounted on adhesive slides. Formalin-fixed, paraffin-embedded (FFPE) tissues are commonly used .

• Antigen Retrieval: Perform heat-induced epitope retrieval with sodium citrate buffer (pH 6.0) for 20 minutes at 80°C .

• Endogenous Peroxidase Blocking: Incubate slides with 3% H₂O₂ in 1X PBS at room temperature for 10 minutes .

• Antibody Dilution: Use anti-CDC27 antibody at a 1:100 dilution (e.g., ab10538) .

• Incubation: Incubate slides with primary antibody at room temperature for 2 hours after blocking .

• Detection System: Use an HRP-conjugated detection system such as Vectastain Elite ABC Kit followed by DAB chromogen development for 10 minutes at room temperature .

• Counterstaining: Counterstain with hematoxylin for 1 minute at room temperature .

• Evaluation Method: Assess CDC27 expression using the histoscore (H-score) system, which considers both staining intensity and percentage of positive cells .

How can I differentiate between nuclear and cytoplasmic CDC27 expression in cancer research?

Differentiating between nuclear and cytoplasmic CDC27 expression is crucial as they may have distinct biological implications:

• Staining Protocol Optimization: Use the standard IHC protocol described above, but pay particular attention to antigen retrieval conditions to preserve both nuclear and cytoplasmic antigens .

• Antibody Selection: Choose antibodies validated for detecting both nuclear and cytoplasmic forms of CDC27, such as those recognizing the C-terminus (amino acids 814-823) .

• Evaluation Method: Apply the histoscore (H-score) system separately for nuclear and cytoplasmic staining:

  • Score intensity as negative (0), weak (1), moderate (2), or strong (3)

  • Calculate H-score = [1 × (% weak staining) + 2 × (% moderate staining) + 3 × (% strong staining)]

  • H-score ranges from 0 to 300

• Classification: Categorize expression as "high" or "low" based on the mean H-score value in your sample set .

• Statistical Analysis: When analyzing correlations with clinical outcomes, evaluate nuclear and cytoplasmic CDC27 expression independently. Research has shown that nuclear CDC27 expression correlates significantly with poorer survival outcomes in rectal adenocarcinoma, while cytoplasmic CDC27 may be associated with tumor progression and distant metastasis .

What approaches can be used to investigate CDC27 mutations and their functional impact?

To study CDC27 mutations and their functional consequences:

• Mutation Identification: Use whole-exome sequencing (WES) to identify variants in CDC27, as demonstrated in studies of conditions like Hemifacial microsomia (HFM) .

• Structural Prediction:

  • Employ PSIPRED (v3.3) and SWISS-MODEL for predictive structural analysis of the variant effects on CDC27 protein structure

  • Use STRING for protein-protein interaction predictions to assess potential disruption of APC/C complex formation

• Functional Validation:

  • Apply CRISPR/Cas9 for gene knockout in model organisms (e.g., zebrafish) to assess phenotypic consequences

  • Use in situ hybridization (ISH) to examine spatiotemporal expression patterns of CDC27 and related genes

  • Perform immunofluorescence with PH3 and TUNEL assays to assess effects on cell proliferation and apoptosis

• Rescue Experiments: Conduct rescue experiments by injecting wild-type CDC27 mRNA into CDC27-mutant models to verify the specificity of observed phenotypes .

• RNA Sequencing: Compare transcriptomes between mutant and control models to identify downstream effects of CDC27 mutations .

How can I assess the phosphorylation status of CDC27 and its impact on APC/C function?

CDC27 phosphorylation is crucial for APC/C regulation. To investigate:

• Phosphorylation Detection:

  • Immunoprecipitate CDC27 from cell lysates using specific antibodies

  • Analyze by Western blotting with phospho-specific antibodies or by observing mobility shifts (phosphorylated CDC27 typically migrates at a higher molecular weight)

• Phosphatase Treatment:

  • After immunoprecipitation of CDC27 (omitting phosphatase inhibitors), treat samples with λ-phosphatase according to manufacturer's protocol (e.g., New England Biolabs)

  • Compare untreated and phosphatase-treated samples by Western blotting to confirm phosphorylation-dependent mobility shifts

• Cell Cycle Analysis:

  • Synchronize cells at different cell cycle stages using standard methods (thymidine block, nocodazole treatment)

  • Analyze CDC27 phosphorylation status throughout the cell cycle to correlate with APC/C activity

• Kinase Inhibition Studies:

  • Treat cells with specific kinase inhibitors (CDK1, PLK1, etc.) to identify kinases responsible for CDC27 phosphorylation

  • Assess the impact on APC/C activity through ubiquitination assays of known APC/C substrates

• Phospho-mimetic and Phospho-dead Mutants:

  • Generate CDC27 constructs with mutations at key phosphorylation sites (Ser/Thr to Ala for phospho-dead; Ser/Thr to Asp/Glu for phospho-mimetic)

  • Express these mutants in CDC27-depleted cells to assess functional consequences on APC/C activity and cell cycle progression

What are common problems encountered in Western blotting with CDC27 antibodies and how can they be resolved?

For best practice, always include positive and negative controls and validate CDC27 detection using alternative methods like immunoprecipitation or mass spectrometry when establishing a new protocol .

How can I optimize CDC27 antibody performance in immunohistochemistry applications?

To optimize CDC27 detection in immunohistochemistry:

• Fixation Optimization: Test different fixation durations (12-24 hours) with 10% neutral-buffered formalin to preserve epitope accessibility while maintaining tissue morphology.

• Antigen Retrieval Comparison: Compare different antigen retrieval methods:

  • Heat-induced epitope retrieval with sodium citrate buffer (pH 6.0)

  • EDTA buffer (pH 8.0)

  • Enzymatic retrieval with proteinase K

• Antibody Titration: Test a range of antibody dilutions (1:50, 1:100, 1:200, 1:500) to determine the optimal concentration that yields specific staining with minimal background .

• Signal Amplification: For weak signals, consider using:

  • Polymer-based detection systems

  • Tyramide signal amplification

  • Extended DAB development time

• Background Reduction: If background is problematic:

  • Extend blocking time (60 minutes)

  • Add 0.1% Triton X-100 to permeabilize tissues more effectively

  • Use animal serum matching the secondary antibody host species

• Control Tissues: Always include:

  • Positive control tissues known to express CDC27

  • Negative controls (omitting primary antibody)

  • Tissue microarrays for standardization across multiple samples

What strategies can address non-specific binding in CDC27 immunoprecipitation experiments?

To minimize non-specific binding in CDC27 immunoprecipitation:

• Pre-clearing Optimization: Pre-clear lysates with protein G-sepharose for 1-2 hours at 4°C before adding CDC27 antibody to remove proteins with non-specific affinity for beads .

• Antibody Selection: Choose monoclonal antibodies when higher specificity is required, or well-characterized polyclonal antibodies with documented specificity .

• Blocking Agents: Add 1-5% BSA or 0.1-0.5% non-ionic detergents (NP-40 or Triton X-100) to reduce non-specific interactions .

• Washing Stringency: Adjust salt concentration in wash buffers:

  • Low stringency: 150 mM NaCl

  • Medium stringency: 250 mM NaCl

  • High stringency: 500 mM NaCl

• Cross-Linking Consideration: For studying weak or transient interactions, consider cross-linking proteins before lysis using formaldehyde or DSP (dithiobis-succinimidyl propionate).

• Two-Step IP: Perform tandem immunoprecipitation by first pulling down with CDC27 antibody, then eluting under mild conditions and re-immunoprecipitating with an antibody against an expected interaction partner.

• Validation: Confirm specificity by performing reciprocal immunoprecipitation or using CDC27 knockdown/knockout cells as negative controls .

How is CDC27 expression analysis being applied in cancer research and what are the clinical implications?

Recent advances in CDC27 expression analysis in cancer research reveal:

• Prognostic Value: Nuclear CDC27, rather than cytoplasmic, serves as an independent prognostic factor in rectal adenocarcinoma, with high expression correlating with poorer survival outcomes .

• Subcellular Distribution Analysis:

  • Nuclear CDC27 H-scores (mean 168.5) significantly exceed cytoplasmic H-scores (mean 95.8)

  • The subcellular distribution carries distinct clinical implications:

    • Cytoplasmic CDC27: Associated with tumor progression and distant metastasis

    • Nuclear CDC27: Strong correlation with survival outcomes

• Predictive Biomarker Potential: Nuclear CDC27 expression may predict responsiveness to postoperative adjuvant chemotherapy in cancer patients, potentially guiding treatment decisions .

• Immune Infiltration Correlation: CDC27 expression levels correlate with CD4+/CD8+ T cell infiltration in tumor microenvironments, suggesting a potential role in immune response modulation in cancer .

• Therapeutic Target Considerations: The finding that CDC27 may drive cell proliferation, tumor growth, epithelial-mesenchymal transition, and metastasis in colorectal and gastric cancers suggests it could be a valuable therapeutic target .

Future directions include developing CDC27-targeting compounds and establishing standardized evaluation methods for subcellular CDC27 expression in clinical pathology.

What role does CDC27 play in genetic disorders and developmental processes?

Emerging research on CDC27's role in genetic disorders reveals:

• Hemifacial Microsomia (HFM): Whole-exome sequencing has identified de novo variants in CDC27 in multiple probands with HFM, a congenital disorder affecting the first and second pharyngeal arches .

• Mutation Characterization:

  • Multiple nonsynonymous SNVs affecting specific domains:

    • c.T1424G:p.M475R mutation altering the TPR repeat domain

    • c.T77C:p.F26S and c.T80C:p.L27P resulting in amino acid changes from leucine to proline and phenylalanine to serine

    • c.T893G:p.L298* stopgain variant

• Structural Impact: Mutations in CDC27 potentially disrupt:

  • Secondary structure elements (helices, strands)

  • Tertiary protein conformation due to altered amino acid properties (e.g., substitution of hydrophobic methionine with basic arginine)

• Developmental Significance: CDC27's role in the APC/C complex suggests that mutations disrupting its function could lead to abnormal cell cycle regulation during critical developmental periods, particularly affecting neural crest cell migration and proliferation .

• Model Systems: CRISPR/Cas9-mediated knockout in zebrafish is being used to investigate CDC27's role in development, with in situ hybridization examining spatiotemporal expression patterns .

This research area presents opportunities for understanding the molecular basis of developmental disorders and potential therapeutic interventions targeting the APC/C pathway.

What emerging techniques can enhance CDC27 research beyond traditional antibody applications?

Advanced methodologies expanding CDC27 research include:

• CRISPR/Cas9 Gene Editing:

  • Generation of CDC27 knockout models to study loss-of-function effects

  • Creation of knock-in models expressing tagged versions of CDC27 for live imaging

  • Introduction of specific mutations to study structure-function relationships

• Proximity-Based Labeling:

  • BioID or TurboID fusions with CDC27 to identify proximal interacting proteins

  • APEX2-mediated proximity labeling to capture transient interactions within the APC/C complex

• High-Resolution Microscopy:

  • Super-resolution microscopy (STORM, PALM) to visualize CDC27 subcellular localization at nanometer resolution

  • Live-cell imaging with fluorescently tagged CDC27 to track dynamics during cell cycle progression

• Mass Spectrometry Applications:

  • Phosphoproteomics to map CDC27 phosphorylation sites throughout the cell cycle

  • Crosslinking mass spectrometry (XL-MS) to define structural constraints within the APC/C complex

• Single-Cell Techniques:

  • Single-cell RNA-seq to analyze CDC27 expression heterogeneity within tissues

  • Single-cell proteomics to correlate CDC27 protein levels with cell cycle status

• Computational Approaches:

  • Molecular dynamics simulations to predict effects of mutations on CDC27 structure

  • Machine learning models to predict functional consequences of CDC27 variants

These emerging techniques promise to provide deeper insights into CDC27 biology beyond what traditional antibody-based methods can achieve alone.