Phospho-CCNE1 (T77) Antibody

What is Phospho-CCNE1 (T77) and why is it significant in cell cycle research?

Phospho-CCNE1 (T77) refers to the phosphorylated form of Cyclin E1 at threonine 77, a critical site involved in cell cycle control. Cyclin E1 belongs to the highly conserved cyclin family whose members exhibit dramatic periodicity in protein abundance throughout the cell cycle. The phosphorylation at T77 is specifically important for regulating the G1/S transition, making it a crucial target for studying cell proliferation mechanisms . Researchers track this phosphorylation to understand the temporal coordination of mitotic events and the regulatory mechanisms that control cell cycle progression.

What are the primary applications for Phospho-CCNE1 (T77) antibodies?

Phospho-CCNE1 (T77) antibodies are employed in various experimental applications:

These applications allow researchers to detect, localize, and quantify the phosphorylated form of Cyclin E1, providing insights into its role in normal and pathological cell division .

How does Phospho-CCNE1 (T77) antibody differ from other Cyclin E1 phospho-specific antibodies?

Phospho-CCNE1 (T77) antibody specifically targets the threonine 77 phosphorylation site, which differs from other phospho-specific antibodies like Phospho-CCNE1 (T395) that target different phosphorylation sites. The T77 site has a unique sequence context (IPTP D) , while the T395 site has a different sequence context (LLTP P) . These different phosphorylation sites may have distinct functional implications in cell cycle regulation. For instance, while both sites are involved in cell cycle control, they may be phosphorylated by different kinases or at different cell cycle stages, providing specific regulatory mechanisms .

What are the optimal sample preparation methods for Phospho-CCNE1 (T77) antibody detection?

For optimal detection of phospho-CCNE1 (T77):

• Cell Lysis: Use lysis buffers containing phosphatase inhibitors to prevent dephosphorylation during sample preparation. Common lysis buffers include RIPA buffer supplemented with sodium orthovanadate, sodium fluoride, and β-glycerophosphate .

• Protein Quantification: Standardize protein concentration using Bradford or BCA assays to ensure consistent loading.

• Sample Processing: For Western blot applications, handle samples at 4°C and add reducing agent (e.g., β-mercaptoethanol) to sample buffer immediately before denaturation.

• Control Samples: Include positive controls such as NIH/3T3 or HepG2 cell lysates, which have been validated to express phosphorylated CCNE1 .

• Phosphorylation Preservation: Snap-freeze samples in liquid nitrogen immediately after collection and process promptly to minimize phosphorylation loss .

How can I optimize Western blot protocols specifically for Phospho-CCNE1 (T77) detection?

Optimizing Western blot for Phospho-CCNE1 (T77) detection requires specific considerations:

• Gel Percentage: Use 10-12% polyacrylamide gels for optimal resolution of the 47-48 kDa CCNE1 protein .

• Transfer Conditions: Employ wet transfer at 100V for 60-90 minutes with methanol-containing transfer buffer to ensure efficient protein transfer.

• Blocking Solution: Use 5% BSA in TBST rather than milk (which contains phosphatases) to preserve phospho-epitopes .

• Antibody Dilution: Start with the manufacturer's recommended dilution range (1:500-1:1000) and optimize as needed .

• Incubation Time: Incubate with primary antibody overnight at 4°C to maximize specific binding.

• Detection System: Enhanced chemiluminescence (ECL) systems provide sensitive detection of phosphorylated proteins. For quantitative analysis, consider fluorescence-based detection systems .

• Expected MW: Look for bands at approximately 47-48 kDa (calculated MW) though observed MW may be slightly higher (~48 kDa) due to post-translational modifications .

What are common troubleshooting steps for weak or non-specific signals?

When encountering issues with Phospho-CCNE1 (T77) antibody detection:

• Weak Signal Problems:

  • Increase antibody concentration (reduce dilution factor)

  • Extend primary antibody incubation time

  • Ensure phosphatase inhibitors are fresh and active

  • Check sample handling procedures to minimize dephosphorylation

  • Increase protein loading amount (typically 30-40 μg of total protein)

• Non-specific Binding Issues:

  • Increase blocking time or BSA concentration

  • Add 0.1% Tween-20 to antibody dilution buffer

  • Perform additional washes between incubation steps

  • Consider using a more specific monoclonal antibody instead of polyclonal

  • Validate with positive control samples like NIH/3T3 or HepG2 cells

• Background Reduction:

  • Increase wash duration and frequency

  • Optimize secondary antibody dilution

  • Pre-adsorb secondary antibodies if cross-reactivity is suspected

How can Phospho-CCNE1 (T77) antibodies be used in cancer research?

Phospho-CCNE1 (T77) antibodies have significant applications in cancer research:

• Biomarker Development: CCNE1 overexpression and phosphorylation status correlate with poor prognosis in various cancers, making phospho-CCNE1 (T77) a potential prognostic biomarker .

• Therapeutic Target Identification: Studying the phosphorylation status of CCNE1 can help identify novel therapeutic targets that disrupt abnormal cell cycle progression in cancer cells .

• Drug Development Research: These antibodies enable researchers to assess the efficacy of cell cycle inhibitors targeting the CDK2-Cyclin E1 complex by measuring changes in T77 phosphorylation .

• Tumor Subtyping: Cancer heterogeneity studies use phospho-CCNE1 (T77) status to characterize different tumor subtypes, particularly in breast, ovarian, and colorectal cancers .

• Immune Infiltration Analysis: Recent research has identified correlations between CCNE1 expression and immune cell infiltration in various cancers, suggesting potential implications for immunotherapy responses .

A pan-cancer analysis revealed that CCNE1 expression positively correlates with cancer-associated immune infiltration levels in breast cancer (BRCA), colon adenocarcinoma (COAD), lung squamous cell carcinoma (LUSC), stomach adenocarcinoma (STAD), and thymoma (THYM) .

What are the considerations for using Phospho-CCNE1 (T77) antibodies in multiplexed immunoassays?

When designing multiplexed assays incorporating Phospho-CCNE1 (T77) antibodies:

• Antibody Compatibility: Ensure primary antibodies are raised in different host species to avoid cross-reactivity when using multiple detection antibodies simultaneously.

• Spectral Overlap: When using fluorescent secondary antibodies, select fluorophores with minimal spectral overlap to avoid bleed-through during imaging or flow cytometry.

• Validation Controls: Include single-stain controls to establish proper compensation settings and eliminate false-positive signals.

• Phosphorylation Dynamics: Consider the temporal dynamics of different phosphorylation events when designing multiplexed phospho-protein assays; some phosphorylation events may be transient or have different kinetics .

• AlphaLISA Technology: For quantitative multiplexed detection, AlphaLISA SureFire Ultra assays offer high sensitivity for phospho-CCNE1 (T77) detection in cellular lysates, requiring only 10 μL sample volume .

• Sequential Detection: For IHC or IF applications with multiple phospho-targets, sequential immunostaining with complete antibody stripping between rounds may be necessary to prevent cross-reactivity.

How do monoclonal and polyclonal Phospho-CCNE1 (T77) antibodies differ in advanced research applications?

The choice between monoclonal and polyclonal antibodies significantly impacts experimental outcomes:

Monoclonal antibodies like CABP1014 offer superior specificity by recognizing a single epitope around the T77 phosphorylation site, making them ideal for quantitative studies. Polyclonal antibodies like ITP1166 may detect the phosphorylated target even when some epitopes are partially masked by protein interactions, offering advantages in certain complex experimental systems .

How do different reactive species (human vs. mouse) influence experimental design when using Phospho-CCNE1 (T77) antibodies?

Species reactivity is a critical consideration when designing experiments:

• Sequence Conservation: The region around T77 in Cyclin E1 shows high conservation across species, but subtle sequence differences may affect antibody binding affinity and specificity.

• Validation Requirements:

  • Human-reactive antibodies (like CABP1014) should be validated using human cell lines such as HepG2 or HT-29 .

  • Mouse-reactive antibodies may be validated using NIH/3T3 cells .

  • For cross-species studies, validation in each species is essential.

• Control Selection: When using mouse models to study human disease relevance, include species-specific positive controls to confirm antibody reactivity in both species.

• Antibody Selection Guide:

  • For human-only studies: Consider antibodies like CABP0013 with high human specificity .

  • For cross-species work: Select antibodies with validated reactivity across target species, such as those reactive to both human and mouse samples .

• Modification Site Recognition: Ensure the antibody recognizes the conserved phosphorylation site across species. For example, the specific sequence around T77 (IPTP D) should be present in both species for cross-reactive antibodies .

What are the considerations for quantitative analysis of Phospho-CCNE1 (T77) levels?

For accurate quantitative analysis of phosphorylation levels:

How can researchers integrate Phospho-CCNE1 (T77) data with other cell cycle markers for comprehensive analysis?

For holistic cell cycle research, integrate phospho-CCNE1 (T77) data with complementary markers:

• Multi-parameter Analysis Framework:

  • Combine phospho-CCNE1 (T77) with CDK2 activity measurements

  • Include additional cyclins (D1, A, B) and their phosphorylated forms

  • Measure cell cycle inhibitors (p21, p27) to assess regulation

  • Integrate DNA content analysis (propidium iodide staining) for cell cycle phase determination

• Technology Integration Approaches:

  • Flow cytometry: Combine phospho-CCNE1 (T77) staining with DNA content analysis

  • Mass cytometry (CyTOF): Simultaneously measure multiple phospho-proteins

  • Multiplexed immunofluorescence: Visualize spatial relationships between phospho-CCNE1 (T77) and other markers

• Systems Biology Considerations:

  • Correlate phospho-CCNE1 (T77) levels with upstream regulators and downstream effectors

  • Use computational modeling to predict cell cycle progression based on multi-parameter data

  • Integrate transcriptomic data to understand regulation at multiple levels

• Cancer-specific Analysis:

  • Correlate phospho-CCNE1 (T77) with immune infiltration metrics

  • CCNE1 expression has been linked to immune infiltration in breast cancer, colon adenocarcinoma, lung squamous cell carcinoma, stomach adenocarcinoma, and thymoma

  • Combine with genomic instability markers to assess cancer progression

How might advances in single-cell analysis techniques impact Phospho-CCNE1 (T77) research?

Emerging single-cell technologies offer new possibilities for phospho-CCNE1 research:

• Single-Cell Phospho-Proteomics: New mass spectrometry approaches enable detection of phosphorylation events at the single-cell level, allowing researchers to study cell-to-cell variability in CCNE1 phosphorylation status.

• Spatial Proteomics: Techniques like imaging mass cytometry can map phospho-CCNE1 (T77) distribution within tissue contexts while preserving spatial information.

• Live-Cell Phosphorylation Sensors: Development of fluorescent biosensors for real-time monitoring of CCNE1 phosphorylation events in living cells.

• Single-Cell Western Blotting: Emerging microfluidic platforms enable Western blot analysis at the single-cell level, potentially revealing heterogeneity in phospho-CCNE1 (T77) levels within cell populations.

• Single-Cell RNA-Protein Correlation: Simultaneous analysis of CCNE1 mRNA and phospho-protein levels can reveal post-transcriptional regulation mechanisms.

These advances will likely enable deeper understanding of the heterogeneity in CCNE1 phosphorylation and its implications for cell cycle regulation in complex tissues and tumors.

What are the implications of CCNE1 phosphorylation for cancer immunotherapy research?

Recent research highlights interesting connections between CCNE1 and immune responses:

• Immune Infiltration Correlation: Pan-cancer analysis has identified positive correlations between CCNE1 expression and immune cell infiltration in multiple cancer types, including breast cancer, colon adenocarcinoma, lung squamous cell carcinoma, stomach adenocarcinoma, and thymoma .

• Potential Immunotherapy Biomarker: CCNE1 phosphorylation status may serve as a predictive biomarker for immunotherapy response, though more research is needed to establish this connection.

• Cell Cycle-Immune System Cross-talk: Phosphorylated CCNE1 may influence antigen presentation or cytokine production, affecting tumor immunogenicity.

• Combination Therapy Approaches: Targeting CCNE1 phosphorylation together with immune checkpoint inhibitors may represent a novel therapeutic strategy.

• Tumor Microenvironment Influence: CCNE1 phosphorylation in tumor cells may impact the recruitment and function of tumor-infiltrating lymphocytes through mechanisms that remain to be fully elucidated.

This emerging research area suggests potential new directions for combining cell cycle-targeted therapies with immunotherapeutic approaches.