CDK1 Monoclonal Antibody

What is CDK1 and what is its biological significance?

CDK1 (Cyclin-dependent kinase 1, also known as CDC2, CDC28A, or P34CDC2) is a key regulatory protein essential for proper progression through the cell cycle, particularly in the control of mitosis. It functions as a serine/threonine kinase that regulates critical cell cycle transitions when complexed with cyclins, especially cyclin B. CDK1 plays a fundamental role in processes such as cell proliferation, DNA replication, and checkpoint control . Recent pan-cancer analyses have identified CDK1 as significantly upregulated in most common cancers and strongly associated with prognosis, indicating its importance in tumorigenesis . CDK1 influences tumor immunity by mediating tumor infiltration of immune-associated cells, with effects varying across tumor types within the tumor microenvironment . Additionally, CDK1 expression correlates positively with tumor mutational burden (TMB) and microsatellite instability (MSI) in certain cancer types, potentially linking it to treatment response assessment .

What applications are CDK1 monoclonal antibodies suitable for?

CDK1 monoclonal antibodies have been validated for multiple research applications:

The applications have been validated using multiple cell lines including HeLa, MCF7, and Jurkat cells, making these antibodies versatile tools for investigating CDK1 expression and activity under different experimental conditions .

How does CDK1 expression vary across different cancer types?

CDK1 is significantly upregulated in most common cancers as confirmed by comprehensive bioinformatic analyses. High expression of CDK1 correlates with poor prognosis across numerous cancer types including adrenocortical carcinoma (ACC, P=7e-08), kidney renal clear cell carcinoma (KIRC, P=0.033), kidney renal papillary cell carcinoma (KIRP, P=0.017), brain lower grade glioma (LGG, P=76e-07), liver hepatocellular carcinoma (LIHC, P=0.00017), lung adenocarcinoma (LUAD, P=2.6e-05), mesothelioma (MESO, P=7.6e-07), pancreatic adenocarcinoma (PAAD, P=6e-04), sarcoma (SARC, P=0.0063), and skin cutaneous melanoma (SKCM, P=0.037) . This pattern makes CDK1 a valuable target for studying cellular proliferation mechanisms across different tumor types.

How can CDK1 monoclonal antibodies be used to study cell cycle regulation?

CDK1 monoclonal antibodies provide powerful tools for investigating cell cycle regulation at multiple levels:

• Protein Expression Analysis: Western blot analysis using CDK1 antibodies can quantify total cellular CDK1 content across different cell cycle phases or in response to treatments.

• Complex Formation Detection: Co-immunoprecipitation with CDK1 antibodies can isolate and analyze CDK1-cyclin complexes, particularly CDK1-cyclin B, which is critical for G2/M transition .

• Activation Status Assessment: Phospho-specific CDK1 antibodies can distinguish between active and inactive forms, helping researchers understand regulatory mechanisms.

• Subcellular Localization Studies: Immunofluorescence with CDK1 antibodies reveals dynamic localization patterns during cell cycle progression, as CDK1 can be found in the cytoplasm, mitochondria, and nucleus .

• Checkpoint Response Evaluation: CDK1 antibodies can track how checkpoint activation affects CDK1 activity, particularly in response to DNA damage or replication stress.

When designing experiments to study cell cycle regulation using CDK1 antibodies, researchers should synchronize cells at specific cell cycle stages to obtain clear temporal patterns of CDK1 expression and activity changes.

What is the relationship between CDK1 expression and cancer patient prognosis?

Pan-cancer analyses have demonstrated that high CDK1 expression significantly correlates with poor prognosis across multiple tumor types. Comprehensive survival analyses reveal:

Interestingly, high CDK1 expression can paradoxically serve as a protective factor for DFS in some colorectal cancer patients, demonstrating the context-dependent nature of CDK1's prognostic value .

How does CDK1 interact with the tumor immune microenvironment?

CDK1 has been identified as a potential modulator of tumor immunity through various mechanisms:

• Immune Cell Infiltration: CDK1 expression levels correlate with the degree of tumor infiltration by immune-associated cells, though this relationship varies across tumor types .

• Correlation with Immune Checkpoints: CDK1 expression may relate to immune checkpoint molecule expression, potentially affecting immunotherapy response.

• Tumor Mutational Burden Connection: CDK1 positively correlates with TMB in multiple cancers including ACC (P=1.01E-06), BLCA (P=7.20E-07), CHOL (P=0.0447), COAD (P=0.0025), HNSC (P=0.0137), KICH (P=0.0036), KIRC (P=0.0048), LAML (P=0.0237), LGG (P=4.13E-16), LUSC (P=1.15E-05), and PAAD (P=1.71E-04) .

• RNA Methylation Regulation: CDK1 expression positively associates with RNA methylation regulatory proteins, potentially affecting post-transcriptional regulation in the tumor microenvironment .

When investigating these relationships, researchers should consider using CDK1 antibodies in conjunction with immune cell markers in multiplex immunohistochemistry or flow cytometry experiments to determine colocalization and potential functional interactions.

What are the optimal protocols for CDK1 antibody usage in Western blot analysis?

For optimal Western blot results with CDK1 monoclonal antibodies, researchers should follow these methodological guidelines:

• Sample Preparation:

  • Harvest cells at 70-80% confluency

  • Lyse cells in appropriate buffer (e.g., RIPA buffer with protease inhibitors)

  • Centrifuge lysates at 10,000g for 10 minutes at 4°C to remove debris

  • Adjust protein concentration across samples (typically 20-50μg total protein)

• Gel Electrophoresis and Transfer:

  • Use 12% SDS-polyacrylamide gels for optimal CDK1 separation (34 kDa)

  • Transfer to nitrocellulose membranes at 100V for 1 hour or 30V overnight at 4°C

• Antibody Incubation:

  • Block membranes with 4-5% BSA or non-fat milk in TBST

  • Dilute primary CDK1 antibody 1:500-1:2000 in blocking buffer

  • Incubate with primary antibody for 1.5 hours at room temperature or overnight at 4°C

  • Wash membranes 3 times with TBST buffer

  • Apply appropriate HRP-conjugated secondary antibody

• Detection and Controls:

  • Use positive control samples such as HeLa, MCF7, or Jurkat cell lysates

  • Include loading controls (β-actin, GAPDH)

  • For CDK1-cyclin B complexes, consider co-immunoprecipitation before Western blotting

How can researchers validate the specificity of their CDK1 monoclonal antibody?

Ensuring antibody specificity is critical for reliable research results. Validation approaches include:

• Knockout/Knockdown Controls:

  • Use CDK1 siRNA/shRNA knockdown samples

  • Compare with negative control siRNA/shRNA

  • Expect significant reduction in band intensity at 34 kDa

• Peptide Competition Assay:

  • Pre-incubate antibody with blocking peptide (such as the immunogen peptide corresponding to amino acids 198-297 of human CDK1)

  • Parallel blots with and without peptide competition

  • Specific signals should be eliminated or significantly reduced

• Cross-Reactivity Testing:

  • Test antibody against recombinant CDK1 alongside related CDKs

  • Ensure minimal cross-reactivity with other CDK family members

• Multiple Antibody Validation:

  • Compare results using antibodies recognizing different epitopes of CDK1

  • Consistent results increase confidence in specificity

• Application-Specific Validation:

  • For immunofluorescence: Confirm subcellular localization patterns (cytoplasm, mitochondrion, nucleus)

  • For immunoprecipitation: Verify pull-down of known CDK1 binding partners like cyclins

What considerations should be made when studying CDK1-cyclin interactions?

When investigating CDK1-cyclin complexes, particularly CDK1-cyclin B, researchers should consider:

• Timing of Analysis:

  • CDK1-cyclin B complexes peak during G2/M phase

  • Synchronize cells appropriately (thymidine block, nocodazole arrest)

• Co-Immunoprecipitation Protocol:

  • Use gentle lysis buffers to preserve protein-protein interactions

  • For CDK1-cyclin B complexes, immunoprecipitate with either CDK1 or cyclin B antibodies

  • Use 0.5-4μg antibody for 200-400μg cell extracts

  • Include appropriate controls (IgG, input)

• Detection Methods:

  • Western blot analysis of immunoprecipitates using antibodies against both CDK1 and cyclin B

  • Consider activity assays to measure functional consequences of complex formation

• Competing Interactions:

  • Consider the presence of CDK inhibitors (CKIs) that may affect complex formation

  • Account for post-translational modifications that regulate binding

• Cyclin Specificity:

  • CDK1 can partner with multiple cyclins beyond cyclin B

  • Design experiments to distinguish between different CDK1-cyclin complexes

How can CDK1 monoclonal antibodies contribute to cancer biomarker development?

CDK1 monoclonal antibodies offer valuable tools for cancer biomarker development based on recent findings:

• Prognostic Marker Assessment:

  • Immunohistochemical analysis of tumor tissues using calibrated CDK1 antibodies

  • Correlation of staining intensity with patient outcomes across multiple cancer types

  • Integration with other markers for improved prognostic models

• Predictive Biomarker Development:

  • Evaluation of CDK1 expression in relation to treatment response

  • Assessment of correlations between CDK1 and TMB/MSI status

  • Potential for predicting response to cell cycle-targeted therapies

• Multi-marker Panels:

  • Combination of CDK1 with other cell cycle regulators

  • Development of immunohistochemical panels for tumor classification

  • Integration with genomic and transcriptomic data

• Liquid Biopsy Applications:

  • Detection of CDK1 in circulating tumor cells

  • Correlation with disease progression or treatment response

Systematic pan-cancer analyses have identified specific tumor types where CDK1 expression most strongly correlates with prognosis, suggesting prioritization of these cancer types for biomarker development efforts .

What are the critical considerations when designing CDK1 inhibition studies?

When designing experiments to study CDK1 inhibition (e.g., with indirubin derivatives), researchers should consider:

• Baseline Expression Assessment:

  • Use CDK1 antibodies to determine baseline expression in cell models

  • Quantify CDK1-cyclin B complex levels before inhibition

• Inhibitor Specificity:

  • Account for potential off-target effects on related kinases

  • Include appropriate controls to distinguish CDK1-specific effects

• Temporal Dynamics:

  • Monitor CDK1 activity at multiple time points after inhibitor treatment

  • Assess both short-term and long-term consequences

• Functional Readouts:

  • Cell cycle analysis by flow cytometry

  • Mitotic index determination

  • Apoptosis assessment

  • Colony formation assays

• Mechanistic Validation:

  • Analyze phosphorylation status of CDK1 substrates

  • Monitor CDK1-cyclin B complex formation after inhibition

  • Combine with genetic approaches (CDK1 knockdown/knockout)

How might CDK1 antibodies be utilized in emerging therapeutic approaches?

CDK1 monoclonal antibodies could contribute to novel therapeutic developments:

• CAR-T and Immunotherapy Approaches:

  • While not yet developed for CDK1, the success of antibody-based CAR-T targeting other kinases (like DCLK1) provides a conceptual framework

  • CDK1 antibodies could help identify patient populations likely to benefit from cell cycle-targeted therapies

• Antibody-Drug Conjugates (ADCs):

  • Potential development of CDK1-targeting ADCs for cancer therapy

  • CDK1 antibodies essential for validating target accessibility

• Combination Therapy Optimization:

  • Use of CDK1 antibodies to monitor pathway modulation during combination treatments

  • Identification of synergistic drug combinations based on CDK1 pathway activity

• Resistance Mechanism Studies:

  • Investigation of CDK1 expression and activity changes in treatment-resistant populations

  • Development of strategies to overcome resistance

• Novel Delivery Systems:

  • Nanoparticle-based delivery of CDK1-targeted therapies

  • Use of CDK1 antibodies to validate target engagement

What novel methodologies might enhance CDK1 research?

Emerging technologies that could advance CDK1 research include:

• Live-Cell Imaging Applications:

  • Development of CDK1 biosensors for real-time activity monitoring

  • CDK1 antibody fragments for live-cell tracking

• Single-Cell Analysis:

  • Application of CDK1 antibodies in single-cell proteomics

  • Correlation with single-cell transcriptomics for multi-omics integration

• High-Content Screening:

  • Use of CDK1 antibodies in automated high-content screening platforms

  • Identification of novel regulators or inhibitors of CDK1 activity

• Spatial Transcriptomics Integration:

  • Combination of CDK1 immunohistochemistry with spatial transcriptomics

  • Mapping of CDK1 activity in the context of tumor microenvironment

• AI and Machine Learning Applications:

  • Development of image analysis algorithms for CDK1 staining pattern recognition

  • Predictive modeling of CDK1 activity based on multi-parametric data