CCNE1 (Ab-395) Antibody

What is CCNE1 and what role does phosphorylation at Threonine 395 play?

CCNE1 (Cyclin E1) is essential for controlling the cell cycle at the G1/S (start) transition, serving as a regulatory subunit of CDK2 . Phosphorylation at Threonine 395 (T395) represents a critical post-translational modification that regulates Cyclin E1 stability and function . This phosphorylation site has been identified as particularly important because it affects ubiquitin-dependent proteolysis of Cyclin E1 mediated by the SCF-Fbxw7 complex . Research has shown that PP2A-B55β phosphatase antagonizes this phosphorylation, thereby promoting Cyclin E1 stability and potentially contributing to its overexpression in cancer cells .

The species reactivity of CCNE1 (Ab-395) Antibody varies by manufacturer:

Always verify the specific reactivity profile of your antibody lot before conducting cross-species experiments .

How should I store and handle the CCNE1 (Ab-395) Antibody?

Proper storage and handling are crucial for maintaining antibody performance:

• Long-term storage: -20°C for up to one year

• Short-term/frequent use: 4°C for up to one month or six months (depending on manufacturer)

• Avoid repeated freeze-thaw cycles as they can degrade antibody quality and performance

• Most formulations contain 50% glycerol in PBS with 0.02% sodium azide at pH 7.2-7.4

Following these guidelines will help preserve antibody specificity and sensitivity throughout your research project .

How does PP2A-B55β regulation of Cyclin E1 phosphorylation at T395 impact cancer research applications?

Research has revealed a critical relationship between PP2A-B55β and Cyclin E1 stability through the regulation of T395 phosphorylation . Studies show that B55β expression correlates strongly with Cyclin E1 protein levels in breast cancer specimens (R²=0.41), surpassing the correlation with CCNE1 gene expression (R²=0.14) . This relationship suggests that:

• PP2A-B55β antagonizes Cyclin E1 proteolysis by protecting it from ubiquitin-dependent degradation

• B55β knockdown reduces Cyclin E1 accumulation at the G1/S boundary while increasing T395 phosphorylation

• B55β expression mirrors Cyclin E1 protein levels during cell cycle progression

For cancer researchers, this understanding provides mechanistic insight into why some tumors overexpress Cyclin E1 protein without corresponding increases in gene expression, suggesting post-translational regulation as a key factor in oncogenesis .

What controls should be included when using phospho-specific CCNE1 (T395) antibodies in experimental design?

When utilizing phospho-specific antibodies targeting CCNE1 T395, comprehensive controls are essential for robust data interpretation:

Incorporating these controls enhances experimental rigor and enables confident interpretation of phosphorylation-specific signals across different experimental conditions .

How can I differentiate between total CCNE1 and phospho-T395 CCNE1 in complex cancer specimens?

Distinguishing total CCNE1 from its phosphorylated form in heterogeneous cancer specimens requires a strategic experimental approach:

• Sequential Immunoblotting: Perform Western blots with phospho-T395 antibody, then strip and reprobe with total CCNE1 antibody to calculate phosphorylation ratio

• Dual Immunohistochemistry: For tissue samples, consider:

  • Serial section staining with phospho-T395 and total CCNE1 antibodies

  • Quantitative analysis of nuclear versus cytoplasmic staining patterns, as phosphorylation may alter subcellular localization

• Phosphatase Inhibitor Optimization: When extracting proteins from tissues, use a comprehensive phosphatase inhibitor cocktail to preserve physiological phosphorylation status

This approach provides more meaningful data than absolute levels alone, particularly when comparing specimens with variable total CCNE1 expression .

What is the relationship between Cyclin E1 T395 phosphorylation and cell cycle progression?

The phosphorylation of Cyclin E1 at T395 serves as a critical regulatory mechanism for cell cycle control:

• T395 phosphorylation increases as cells progress from G1 into S phase, marking Cyclin E1 for ubiquitin-mediated degradation by SCF-Fbxw7

• PP2A-B55β expression closely mirrors Cyclin E1 protein levels, peaking at the G1/S boundary and decreasing as cells progress into S phase

• Temporal regulation pattern:

  • Low T395 phosphorylation → high Cyclin E1 stability → G1/S transition promotion

  • High T395 phosphorylation → increased Cyclin E1 degradation → S phase progression

• Experimental data shows B55β knockdown significantly reduces Cyclin E1 accumulation at the G1/S boundary while simultaneously increasing T395 phosphorylation

This relationship has significant implications for cancer research, as dysregulation of this phosphorylation event may contribute to abnormal cell proliferation through improper Cyclin E1 accumulation or degradation .

What is the optimal protocol for using CCNE1 (Ab-395) Antibody in Western blotting?

For optimal Western blotting results with CCNE1 (Ab-395) antibody:

Sample Preparation:

• Extract total protein using RIPA buffer supplemented with phosphatase inhibitors

• Quantify and normalize protein loading (30-50μg recommended)

• Denature samples in Laemmli buffer at 95°C for 5 minutes

Gel Electrophoresis and Transfer:

• Separate proteins on 10-12% SDS-PAGE gels (Cyclin E1 predicted MW: 47 kDa)

• Transfer to PVDF membrane (0.45μm pore size) at 100V for 60-90 minutes

Antibody Incubation:

• Block membrane with 5% BSA in TBST for 1 hour at room temperature

• Incubate with primary antibody at 1:500-1:1000 dilution overnight at 4°C

• Wash 3×10 minutes with TBST

• Incubate with HRP-conjugated secondary antibody (1:5000) for 1 hour at room temperature

• Wash 3×10 minutes with TBST

Detection and Controls:

• Develop using ECL substrate and appropriate imaging system

• Include phosphopeptide competition controls to confirm specificity

• Consider using paclitaxel-treated HeLa cells (1μM, 60 minutes) as a positive control

This protocol has been optimized based on validated approaches and will provide specific detection of phosphorylated Cyclin E1 at T395 .

How should I optimize immunohistochemistry protocols for CCNE1 (Ab-395) Antibody?

Successful immunohistochemistry with CCNE1 (Ab-395) antibody requires careful optimization:

Tissue Preparation:

• Fix tissues in 10% neutral buffered formalin (24-48 hours)

• Process and embed in paraffin following standard protocols

• Section at 4-5μm thickness

Antigen Retrieval (Critical Step):

• Heat-induced epitope retrieval in citrate buffer (pH 6.0) for 20 minutes

• Allow slides to cool in buffer for 20 minutes

Staining Protocol:

• Block endogenous peroxidase with 3% H₂O₂ (10 minutes)

• Block non-specific binding with 5% normal goat serum (1 hour)

• Apply primary antibody at 1:50-1:200 dilution (overnight at 4°C)

• Wash thoroughly with PBS (3×5 minutes)

• Apply appropriate HRP-conjugated secondary antibody (30-60 minutes)

• Develop with DAB and counterstain with hematoxylin

Validation Controls:

• Include positive control tissue (human colon carcinoma or placenta)

• Run negative controls (primary antibody omission and isotype control)

• Include phosphopeptide competition control on serial sections

Careful optimization of these parameters will ensure specific nuclear staining pattern characteristic of phosphorylated Cyclin E1 .

What are the best approaches for quantifying CCNE1 phosphorylation levels in experimental samples?

Accurate quantification of CCNE1 phosphorylation requires careful consideration of several methodological approaches:

Western Blot Quantification:

• Use dual detection of phospho-T395 and total CCNE1

• Analyze band intensities with appropriate software (ImageJ, Image Lab)

• Calculate phosphorylation ratio: (phospho-CCNE1/total CCNE1)

• Normalize to loading control (β-actin, GAPDH)

Immunohistochemistry Quantification:

• Use digital pathology software for automated scoring

• Assess:

  • Percentage of positive cells

  • Staining intensity (0-3+ scale)

  • H-score calculation (% positive cells × intensity)

• Compare parallel sections stained for phospho and total protein

Flow Cytometry Approach:

• Fix cells in 4% paraformaldehyde

• Permeabilize with 0.1% Triton X-100

• Dual stain for phospho-CCNE1 and DNA content

• Analyze phosphorylation status across cell cycle phases

ELISA-Based Quantification:

• Develop sandwich ELISA with capture antibody against total CCNE1

• Detect with phospho-T395 specific antibody

• Generate standard curve using recombinant phosphorylated protein

The optimal approach depends on your experimental design, sample type, and required sensitivity/specificity balance .

How can I effectively use CCNE1 (Ab-395) Antibody in cell cycle analysis experiments?

For effective cell cycle analysis using CCNE1 (Ab-395) antibody:

Synchronization Strategies:

• Double thymidine block for G1/S boundary arrest

• Nocodazole treatment for mitotic arrest

• Serum starvation for G0/G1 arrest

Multiparameter Analysis Protocol:

• Fix cells in 70% ethanol (overnight at -20°C)

• Permeabilize with 0.25% Triton X-100 (10 minutes)

• Block with 3% BSA (30 minutes)

• Incubate with phospho-T395 CCNE1 antibody (1:100, 2 hours)

• Add fluorophore-conjugated secondary antibody (1 hour)

• Counterstain DNA with propidium iodide or DAPI

• Analyze by flow cytometry or high-content imaging

Experimental Design Considerations:

• Include B55β knockdown as a phosphorylation modulator

• Compare synchronized versus asynchronous populations

• Correlate phospho-T395 signal with cell cycle markers:

  • Cyclin A (S phase)

  • Cyclin B1 (G2/M phase)

  • Ki-67 (proliferating cells)

This approach enables precise correlation between CCNE1 phosphorylation status and cell cycle progression, particularly at the critical G1/S transition point .

What are common issues encountered with CCNE1 (Ab-395) Antibody and how can they be resolved?

Researchers may encounter several challenges when working with phospho-specific antibodies like CCNE1 (Ab-395):

For phospho-T395 specific detection, researchers should particularly focus on maintaining phosphorylation status throughout sample preparation by using phosphatase inhibitors and keeping samples cold .

How can I distinguish true biological findings from technical artifacts when studying CCNE1 phosphorylation?

Distinguishing biological significance from technical artifacts requires systematic experimental design and controls:

Validation Approaches:

• Biological Validation:

  • Modulate phosphorylation with known regulators (PP2A-B55β knockdown/overexpression)

  • Compare cell cycle-synchronized populations

  • Correlate with functional outcomes (cell proliferation, cell cycle progression)

• Technical Validation:

  • Reproduce findings with alternative antibody clones

  • Confirm with orthogonal methods (mass spectrometry)

  • Validate with phospho-null mutants (T395A) and phospho-mimetic mutants (T395E)

• Quantitative Assessment:

  • Establish dose-response relationships

  • Perform time-course analyses

  • Quantify signal-to-noise ratios across experiments

• Control Implementation:

  • Use phosphopeptide competition to confirm specificity

  • Include biologically relevant positive controls (paclitaxel-treated HeLa cells)

  • Compare signals before/after phosphatase treatment

What considerations are important when interpreting CCNE1 phosphorylation data in cancer research?

When interpreting CCNE1 phosphorylation data in cancer contexts, several factors require careful consideration:

Biological Context Factors:

• Tissue-Specific Regulation: Phosphorylation patterns may vary across tissue types and cancer subtypes

• Correlation with Outcomes: Assess how phosphorylation status correlates with:

  • Proliferation indices

  • Patient survival data

  • Treatment response

Technical Interpretation Considerations:

• Signal Quantification: Normalize phospho-signal to total CCNE1 rather than analyzing absolute levels

• Heterogeneity Assessment: Consider tumor heterogeneity and stromal contamination in tissue samples

• Threshold Definition: Establish biologically meaningful thresholds for "high" versus "low" phosphorylation

Mechanistic Interpretation Framework:

• Pathway Integration: Connect T395 phosphorylation to:

  • PP2A-B55β expression levels

  • SCF-Fbxw7 activity

  • CDK2 activation status

• Functional Correlation: Link phosphorylation data to:

  • Cell cycle distribution

  • Genomic instability markers

  • Therapeutic vulnerability

Translational Considerations:

• Biomarker Potential: Evaluate whether phospho-T395 CCNE1 could serve as a:

  • Diagnostic biomarker

  • Prognostic indicator

  • Predictive marker for specific therapies

This contextual approach to data interpretation enhances the translational relevance of CCNE1 phosphorylation findings in cancer research .

How should I design experiments to study the relationship between PP2A-B55β and CCNE1 T395 phosphorylation?

Based on published research, the following experimental design would effectively investigate the PP2A-B55β/CCNE1 relationship:

Genetic Manipulation Approaches:

• siRNA-Mediated Knockdown:

  • Target B55β in relevant cell lines

  • Evaluate effects on:

    • Total CCNE1 levels

    • T395 phosphorylation status

    • Cell cycle progression

• Ectopic Expression:

  • Overexpress B55β in cancer cell lines

  • Assess CCNE1 stability and phosphorylation

  • Monitor effects on cell proliferation

Biochemical Analysis Design:

• Protein-Protein Interaction:

  • Co-immunoprecipitation of PP2A-B55β and CCNE1

  • Proximity ligation assays in intact cells

  • Mapping interaction domains

• Phosphatase Activity Assays:

  • In vitro dephosphorylation of CCNE1 peptides

  • Phosphatase inhibitor studies

  • Kinetic analysis of dephosphorylation rates

Cell Cycle Synchronization Experiments:

• Temporal Analysis:

  • Synchronize cells at G1/S boundary

  • Release and collect timepoints

  • Analyze B55β expression, CCNE1 levels, and T395 phosphorylation

  • Correlate with cell cycle markers (Cyclin A, Cyclin B1)

• Single-Cell Analysis:

  • Combine phospho-flow cytometry with cell cycle analysis

  • Correlate T395 phosphorylation with DNA content

  • Assess cell-to-cell variability

This comprehensive experimental approach would provide mechanistic insight into how PP2A-B55β regulates CCNE1 stability through T395 dephosphorylation .

What are the most significant research applications for CCNE1 (Ab-395) Antibody in current literature?

The CCNE1 (Ab-395) Antibody has emerged as a valuable tool for investigating several critical aspects of cell cycle regulation and cancer biology:

• Cell Cycle Regulation Studies: Enables precise tracking of phosphorylation-dependent regulation of Cyclin E1 at the G1/S transition

• Cancer Research Applications:

  • Investigating mechanisms of Cyclin E1 overexpression in tumors

  • Studying post-translational regulation that may contribute to oncogenesis

  • Exploring the relationship between PP2A-B55β expression and Cyclin E1 stability in cancer specimens

• Phosphorylation-Dependent Proteolysis: Examining how T395 phosphorylation affects SCF-Fbxw7-mediated ubiquitination and degradation of Cyclin E1

• Phosphatase Network Mapping: Investigating the counterbalance between kinase and phosphatase activities that regulate Cyclin E1 stability and function

• Biomarker Development: Potential use in distinguishing tumors with post-translational versus transcriptional Cyclin E1 upregulation