Phospho-CDC16 (S560) Antibody

What is CDC16 and what is its biological significance?

CDC16 (also known as ANAPC6) is a critical component of the anaphase-promoting complex/cyclosome (APC/C), which functions as a multi-subunit E3 ubiquitin ligase essential for cell cycle progression. CDC16 serves as one of the core subunits of the APC/C along with CDC27 . The APC/C complex primarily functions in the ubiquitination of cyclin B, resulting in cyclin B/Cdk complex degradation, which is essential for cells to exit mitosis and enter G1 phase . As part of the ubiquitin-proteasome pathway, CDC16 plays a crucial role in the tightly regulated process of cell division.

CDC16 has a calculated molecular weight of approximately 71.6 kDa and belongs to the APC6/CDC16 family of proteins . In humans, the CDC16 gene encodes a protein that is also referred to as APC6, CDC16 homolog, CDC16Hs, or Cyclosome subunit 6 in the scientific literature .

What is the significance of S560 phosphorylation on CDC16?

The serine 560 (S560) residue represents a critical phosphorylation site on the CDC16 protein. Phosphorylation at this specific residue is believed to modulate APC/C activity during mitosis . While the precise functional consequences of S560 phosphorylation have not been fully characterized, previous studies have identified numerous phosphorylation sites on APC/C isolated from human mitotic cells, suggesting their importance in APC/C regulation .

Phosphorylation of APC/C components, including CDC16, appears to be a key mechanism for regulating APC/C assembly and activity during cell cycle progression. Similar to how CDC20 phosphorylation has been found to inhibit binding of CDC20 to APC/C and recruitment of UBE2S , CDC16 phosphorylation likely contributes to the dynamic regulation of APC/C function during mitosis.

How is the Phospho-CDC16 (S560) Antibody generated?

Phospho-CDC16 (S560) antibodies are typically generated using synthetic peptides derived from the region surrounding the S560 phosphorylation site of human CDC16. According to multiple manufacturers, the immunogen used is a synthesized peptide derived from human CDC16 around the phosphorylation site of S560 .

The antibody production process generally follows these steps:

• Generation of a synthetic phosphopeptide corresponding to the region around S560

• Immunization of rabbits with the phosphopeptide conjugated to a carrier protein

• Collection of antiserum from immunized rabbits

• Affinity purification using epitope-specific immunogen chromatography

The resulting antibodies are polyclonal in nature and specifically recognize CDC16 protein only when phosphorylated at S560 .

What are the key specifications of commercial Phospho-CDC16 (S560) Antibodies?

Below is a comparative table of technical specifications compiled from multiple commercial sources:

What are the recommended experimental conditions for different applications?

The following table provides recommended dilutions and conditions for various applications based on manufacturer guidelines:

These dilutions serve as starting points for optimization, and researchers should determine the optimal working concentration for their specific experimental conditions .

How can researchers validate the specificity of Phospho-CDC16 (S560) Antibody?

Validation of phospho-specificity is critical when working with phosphorylation-state specific antibodies. Several approaches are recommended:

• Blocking peptide competition: Pre-incubation of the antibody with the phosphopeptide immunogen should abolish the signal in all applications. Multiple manufacturers demonstrate this validation approach in their technical documentation .

• Phospho-ELISA: Comparing antibody reactivity between phosphorylated and non-phosphorylated peptides using ELISA can quantitatively demonstrate specificity .

• Phosphatase treatment: Treating samples with lambda phosphatase prior to immunoblotting should eliminate the signal if the antibody is truly phospho-specific.

• Multiple application validation: Confirming specificity across different applications (WB, IHC, IF) strengthens confidence in antibody specificity .

The images provided by manufacturers typically demonstrate these validation approaches, showing signal with phosphorylated samples and absence of signal when blocked with the specific phosphopeptide .

What are the key considerations for Western blot detection of phosphorylated CDC16?

When analyzing phosphorylated CDC16 by Western blot, researchers should consider several technical factors:

• Gel composition: To properly resolve the phosphorylated forms of CDC16, a 10% polyacrylamide gel containing 0.13% bisacrylamide is recommended . This specific composition is crucial for separating phosphorylated and non-phosphorylated forms.

• Sample preparation: Samples should be prepared with phosphatase inhibitors to prevent dephosphorylation during extraction. Common inhibitors include sodium fluoride, sodium orthovanadate, and phosphatase inhibitor cocktails.

• Loading controls: When comparing phosphorylation levels across samples, total CDC16 levels should be assessed in parallel using a non-phospho-specific CDC16 antibody . This allows normalization to total protein levels.

• Positive controls: Cell lysates from mitotic cells (e.g., nocodazole-arrested) can serve as positive controls for CDC16 phosphorylation, as APC/C components are known to be highly phosphorylated during mitosis .

• Signal detection: Enhanced chemiluminescence (ECL) is commonly used for detection, with exposure times optimized to avoid signal saturation, especially important for quantitative analyses.

How can Phospho-CDC16 (S560) Antibody be used in cell cycle research?

Phospho-CDC16 (S560) Antibody can be a valuable tool for investigating cell cycle regulation, particularly during mitosis:

• Cell synchronization studies: Researchers can synchronize cells at different cell cycle stages (e.g., using thymidine block, nocodazole, or hydroxyurea ) and analyze CDC16 phosphorylation status across the cell cycle.

• Kinase inhibition experiments: Treatment with specific kinase inhibitors can help identify the kinases responsible for CDC16 S560 phosphorylation. Changes in phosphorylation levels can be monitored by Western blot or immunofluorescence.

• Co-immunoprecipitation: Phospho-CDC16 (S560) Antibody can be used to immunoprecipitate phosphorylated CDC16 and analyze its interaction partners, providing insights into how phosphorylation affects APC/C complex formation.

• Fluorescence microscopy: Immunofluorescence with Phospho-CDC16 (S560) Antibody can reveal the subcellular localization of phosphorylated CDC16 throughout the cell cycle, particularly in relation to spindle formation and chromosome segregation .

• Cell-based ELISA: Qualitative determination of CDC16 phosphorylation levels can be achieved using cell-based ELISA formats, which allow normalization using various methods including GAPDH expression, crystal violet staining, or total CDC16 levels .

What normalization approaches are recommended for cell-based assays?

For cell-based assays using Phospho-CDC16 (S560) Antibody, several normalization methods are recommended:

• Internal control normalization: Anti-GAPDH antibody can serve as an internal positive control for normalizing target absorbance values in ELISA-based assays .

• Cell density normalization: Following colorimetric measurements, crystal violet whole-cell staining can determine cell density, allowing absorbance values to be normalized to cell amounts to adjust for plating differences .

• Total protein normalization: Anti-CDC16 antibody (non-phospho-specific) can be used for normalization purposes. The absorbance values obtained for non-phosphorylated CDC16 can normalize the values for phosphorylated CDC16 .

• Housekeeping protein controls: When performing Western blot analysis, normalization to housekeeping proteins such as GAPDH, β-actin, or α-tubulin is recommended for comparing phosphorylation levels across different samples.

How does CDC16 phosphorylation integrate with broader APC/C regulation mechanisms?

CDC16 phosphorylation represents one component of the complex regulatory network controlling APC/C activity. Advanced research questions include:

• Phosphorylation cascades: Evidence suggests that cyclin-dependent kinases (CDKs) like Cdc28 are involved in phosphorylating APC/C components . How does S560 phosphorylation fit within these broader phosphorylation cascades?

• Temporal coordination: How is CDC16 S560 phosphorylation temporally coordinated with other post-translational modifications of APC/C components during mitotic progression?

• Structural consequences: How does S560 phosphorylation affect the three-dimensional structure of CDC16 and its integration within the APC/C complex?

• Feedback regulation: Is CDC16 phosphorylation part of feedback mechanisms that fine-tune APC/C activity in response to mitotic progression or cellular stresses?

Methodologically, addressing these questions may require combining Phospho-CDC16 (S560) Antibody with structural biology approaches, mass spectrometry, and real-time imaging of APC/C activity in living cells.

What are the emerging applications of Phospho-CDC16 (S560) Antibody in disease research?

Given the critical role of APC/C in cell cycle regulation, Phospho-CDC16 (S560) Antibody has potential applications in disease research:

• Cancer research: Dysregulation of the APC/C is implicated in several cancers. Researchers can use Phospho-CDC16 (S560) Antibody to investigate whether aberrant CDC16 phosphorylation contributes to malignant transformation or tumor progression.

• Neurodegenerative diseases: Post-mitotic neurons rely on APC/C function for processes beyond cell division. Investigating CDC16 phosphorylation in neuronal contexts may provide insights into neurodegenerative conditions.

• Development and differentiation: APC/C plays roles in cellular differentiation. Tracking CDC16 phosphorylation during development could illuminate how cell cycle machinery is repurposed during differentiation.

• Drug development: Phospho-CDC16 (S560) Antibody can be used to screen compounds that modulate APC/C activity, potentially identifying novel cell cycle-targeting therapeutics.

• Biomarker discovery: Analyzing CDC16 phosphorylation patterns across disease states may reveal potential diagnostic or prognostic biomarkers.

How can phospho-proteomics approaches complement antibody-based detection of CDC16 phosphorylation?

While antibody-based detection remains valuable, complementary phospho-proteomics approaches can provide broader insights:

• Global phosphorylation analysis: Mass spectrometry-based phospho-proteomics can identify multiple phosphorylation sites on CDC16 simultaneously, placing S560 phosphorylation in a broader context.

• Phosphorylation dynamics: Using SILAC (Stable Isotope Labeling with Amino acids in Cell culture) or TMT (Tandem Mass Tag) labeling combined with mass spectrometry allows quantitative analysis of phosphorylation dynamics across conditions or time points.

• Kinase prediction algorithms: Computational approaches can predict potential kinases responsible for S560 phosphorylation based on consensus sequences, which can then be validated experimentally.

• System-level integration: Phospho-proteomics data can integrate CDC16 phosphorylation into system-level models of cell cycle regulation, providing a more comprehensive understanding.

• Multiplexed detection: Bio-Plex phosphoprotein assays, which utilize phosphorylation state-specific antibodies, enable researchers to simultaneously measure multiple phosphorylation events, placing CDC16 phosphorylation in the context of broader signaling networks .