Phospho-CCNB1 (S147) Antibody

What is CCNB1 and why is phosphorylation at S147 significant?

Cyclin B1 (CCNB1) is a regulatory protein involved in mitosis that complexes with CDK1 (p34/cdc2) to form the maturation-promoting factor crucial for cell cycle progression . Phosphorylation at Serine 147 represents one of several key post-translational modifications that regulate Cyclin B1 activity during the cell cycle. The phosphorylation at S147 is part of a broader network of phosphorylation events that orchestrate proper cell division timing and checkpoint regulation . Notably, while S126 and S128 are known to be phosphorylated by CDK1, PLK1, and MAPK1 , the specific kinases responsible for S147 phosphorylation require further characterization in different cellular contexts.

What are the optimal storage conditions for maintaining Phospho-CCNB1 (S147) antibody activity?

For long-term storage, maintain the antibody at -20°C for up to one year. For frequent use over shorter periods (up to one month), storage at 4°C is recommended . The antibody is typically supplied in a buffer containing PBS with 50% glycerol, 0.5% BSA, and 0.02% sodium azide . Critically, avoid repeated freeze-thaw cycles as they significantly degrade antibody performance and specificity . If multiple uses are anticipated, consider preparing working aliquots before freezing to minimize freeze-thaw damage to the primary stock.

What are the validated applications for Phospho-CCNB1 (S147) antibodies?

The Phospho-CCNB1 (S147) antibody has been validated for multiple experimental applications including:

The optimal working dilution should be determined empirically for each experimental setup and sample type . For most mammalian research models, this antibody shows reactivity with human, mouse, and rat samples .

How can researchers validate antibody specificity for phosphorylated S147?

A rigorous validation approach should include:

• Positive and negative controls: Include samples with known phosphorylation status (e.g., cell cycle synchronized samples).

• Phosphatase treatment: Compare results before and after treatment with lambda phosphatase to confirm phospho-specificity.

• Peptide competition: Pre-incubation of the antibody with phosphorylated and non-phosphorylated peptides should show differential blocking effects.

• Correlation with cellular conditions: Phosphorylation levels should correspond with expected cell cycle phases when CCNB1-S147 is phosphorylated.

The antibody exhibits high specificity with no reported cross-reactivity to other proteins, though cross-reactivity with other phosphorylation sites on CCNB1 should be experimentally excluded .

What methodologies can be used to study dynamic phosphorylation of CCNB1 during cell cycle progression?

To investigate temporal dynamics of S147 phosphorylation:

• Synchronized cell populations: Use techniques like double thymidine block or nocodazole treatment to synchronize cells at specific cell cycle stages.

• Time-course experiments: Collect samples at defined intervals following synchronization release.

• Quantitative immunoblotting: Combine with total CCNB1 detection to calculate the phosphorylation ratio.

• Immunofluorescence microscopy: For spatial and temporal resolution within individual cells.

• Phosphoproteomics: For larger-scale analysis, mass spectrometry-based approaches can validate and quantify S147 phosphorylation in complex samples.

Research has identified approximately 4,000 phosphopeptide sequences in related studies, with 680 phosphorylated at CDK1 consensus sequences (S/T*-P), providing context for CCNB1 phosphorylation within the broader phosphoproteome .

How does S147 phosphorylation interact with other post-translational modifications of CCNB1?

CCNB1 undergoes multiple post-translational modifications including phosphorylation at several sites (T6, S9, S35, S69, S95, S116, S126, S128, and S147), acetylation (K25, K73), ubiquitination (K25, K36, K51), and sumoylation (K111) . These modifications create a complex regulatory network that fine-tunes CCNB1 function throughout the cell cycle. When designing experiments to study S147 phosphorylation:

• Consider potential crosstalk between S147 phosphorylation and modifications at proximal sites.

• Investigate hierarchical phosphorylation patterns to determine if S147 phosphorylation depends on prior modifications.

• Examine modification-specific protein interactions using techniques like proximity ligation assays or immunoprecipitation followed by mass spectrometry.

Understanding these relationships requires systematic mutational analysis (e.g., phosphomimetic and phosphodeficient mutants) coupled with functional assays to assess cell cycle progression effects.

What are the experimental considerations when using phospho-specific antibodies in cells with genetic or pharmacological perturbations?

When using Phospho-CCNB1 (S147) antibodies in perturbed systems:

• Signal validation: Confirm that signal changes truly reflect phosphorylation changes rather than alterations in total protein levels by always normalizing to total CCNB1 expression.

• Kinase inhibition studies: When using CDK inhibitors or other kinase inhibitors, consider both on-target and off-target effects that might indirectly affect S147 phosphorylation.

• Genetic perturbations: In knockout or knockdown studies, assess possible compensatory mechanisms that might maintain phosphorylation through alternative kinases.

• Temporal considerations: Establish appropriate timepoints for analysis based on the kinetics of your perturbation and the cell cycle phase you're investigating.

Recent research approaches have employed specific mutants (like clb2-pp) that affect protein interactions and phosphorylation patterns, revealing that phosphorylation at CDK1 consensus sites may depend on specific binding pockets .

How can phosphoproteomics approaches complement antibody-based detection of CCNB1-S147 phosphorylation?

Mass spectrometry-based phosphoproteomics offers several advantages:

• Unbiased detection: Identifies all phosphorylation sites on CCNB1 simultaneously without reliance on site-specific antibodies.

• Quantitative analysis: Provides stoichiometric information about the fraction of CCNB1 phosphorylated at S147 relative to the total protein.

• Discovery of novel sites: May reveal previously uncharacterized phosphorylation sites that interact with S147.

• Pathway integration: Places S147 phosphorylation in context with broader cell cycle-related phosphorylation events.

Implementation strategies include:

• Enrichment of phosphopeptides using titanium dioxide or immobilized metal affinity chromatography before MS analysis

• SILAC or TMT labeling for quantitative comparison across experimental conditions

• Targeted approaches like selected reaction monitoring for enhanced sensitivity

Research has demonstrated the utility of these approaches by identifying hundreds of CDK1 substrate phosphorylation sites, including those that demonstrate differential phosphorylation in wild-type versus mutant conditions .

What are common technical challenges when working with Phospho-CCNB1 (S147) antibodies?

When working with the Phospho-CCNB1 (S147) antibody, careful optimization of washing steps and incubation times is critical for maintaining specificity while achieving adequate sensitivity . The recommended starting dilutions (ELISA: 1:10000, IHC: 1:100-1:300, WB: 1:500-1:2000) should be systematically titrated for each experimental system .

How should researchers interpret data discrepancies between different detection methods for CCNB1-S147 phosphorylation?

When facing inconsistent results across different methods:

• Consider method-specific limitations: Western blotting detects denatured proteins, while IP-based methods preserve native conformations that might influence antibody recognition.

• Evaluate temporal dynamics: Different methods have different temporal resolution capabilities - single-cell techniques may reveal heterogeneity masked in population-based assays.

• Assess technical variables: Fixation methods for IHC/IF may differentially preserve phospho-epitopes compared to rapid lysis for Western blotting.

• Validate with orthogonal approaches: Complement antibody-based detection with mass spectrometry or functional assays (e.g., kinase assays).

For conclusive interpretation, triangulate findings using multiple detection methods and correlate with functional outcomes relevant to cell cycle progression or other CCNB1-regulated processes.

What considerations should guide experimental design when studying CCNB1-S147 phosphorylation in disease models?

When investigating pathological contexts:

• Sample handling: Phosphorylation status can change rapidly ex vivo - standardize time from collection to fixation/lysis.

• Appropriate controls: Include matched normal tissues or cells for comparative analysis.

• Cell cycle normalization: Account for differences in proliferation rates between normal and disease samples.

• Therapeutic implications: Consider how treatments might affect phosphorylation directly or indirectly.

• Correlation with outcomes: Link phosphorylation patterns to disease progression, therapeutic response, or patient outcomes.

For cancer research particularly, consider how CCNB1-S147 phosphorylation might correlate with cell cycle dysregulation and therapeutic vulnerabilities in specific tumor types.

What emerging technologies might enhance the study of site-specific CCNB1 phosphorylation?

Several cutting-edge approaches show promise for advancing research in this area:

• Biosensors: Developing FRET-based sensors for real-time monitoring of S147 phosphorylation in living cells.

• Optogenetic tools: Light-controlled kinase or phosphatase systems to manipulate S147 phosphorylation with precise spatial and temporal control.

• CRISPR-based approaches: Precise genome editing to create endogenous tags or phospho-mutants of CCNB1.

• Single-cell phosphoproteomics: Emerging methods for measuring phosphorylation events in individual cells to capture heterogeneity.

• Cryo-EM structural studies: Visualizing conformational changes induced by S147 phosphorylation and their impact on protein-protein interactions.

These technologies will help resolve questions about the precise timing and functional consequences of S147 phosphorylation during normal cell cycle and in disease states.

How might understanding CCNB1-S147 phosphorylation contribute to therapeutic development?

The phosphorylation status of CCNB1 at S147 and other sites may offer:

• Biomarker potential: As indicators of cell cycle dysregulation in cancer or other proliferative disorders.

• Target validation: Helping to assess the on-target effects of CDK inhibitors or other cell cycle-targeting drugs.

• Resistance mechanisms: Elucidating how alterations in phosphorylation patterns might contribute to therapeutic resistance.

• Combination strategies: Informing rational design of drug combinations that target complementary cell cycle regulatory mechanisms.

Comprehensive phosphorylation profiling, including S147 status, could eventually guide personalized treatment selection for patients with cell cycle-driven diseases.