CKS2 Antibody

What is CKS2 and what cellular functions does it regulate?

CKS2 (CDC28 protein kinase regulatory subunit 2) is a 10 kDa protein that binds to the catalytic subunit of cyclin-dependent kinases (CDKs) and is essential for their biological function . It plays a vital role in regulating the cell cycle, particularly in somatic cell division and early embryonic development . CKS2 functions as a crucial regulator of cell cycle progression by interacting with CDKs to modulate their activity during critical phases of the cell cycle. Research indicates that CKS2 primarily influences cell division, DNA replication, and cell cycle control pathways based on functional enrichment analyses .

How do CKS2 antibodies differ in their applications for research?

CKS2 antibodies vary in their host species, clonality, and validated applications, which affects their utility in different experimental contexts:

Researchers should select antibodies based on their specific experimental requirements, considering factors such as the detection method, sample type, and need for specificity versus broader epitope recognition .

What methodological approaches are used to study CKS2 expression in tissue samples?

Immunohistochemistry (IHC) is the predominant method for evaluating CKS2 protein expression in tissue samples. The staining protocol typically involves:

• Sample preparation: Formalin-fixed, paraffin-embedded tissue sections

• Antigen retrieval: Heat-mediated antigen retrieval with citrate buffer pH 6

• Primary antibody incubation: Typically at dilutions of 1/50 to 1/100

• Detection system: HRP secondary antibody and DAB treatment

• Quantification: Scoring based on both staining intensity (0-3) and fraction of positive cells (0-4)

Scoring methods incorporate both the intensity of staining and the percentage of positive cells, with the final score calculated as: staining score = staining intensity score × fraction of positive cell score. This approach enables semi-quantitative assessment of CKS2 expression levels across different tissue samples .

How is CKS2 expression associated with cancer prognosis, particularly in lung adenocarcinoma?

CKS2 overexpression demonstrates significant correlation with poor prognosis in lung adenocarcinoma (LUAD) patients. Analysis of multiple patient cohorts from TCGA, GEO, and independent clinical samples reveals that high CKS2 expression is associated with:

What molecular mechanisms explain CKS2's role in cancer progression?

CKS2 contributes to cancer progression through multiple mechanistic pathways:

• Cell cycle dysregulation: CKS2 binding to cyclin-dependent kinase 2 (CDK2) confers partial resistance to inhibitory tyrosine phosphorylation mediated by the intra–S-phase checkpoint, allowing cancer cells to continue DNA replication despite replicative stress .

• Evasion of cell cycle checkpoints: Overexpression of CKS2 leads to override of the intra–S-phase checkpoint that normally blocks DNA replication in response to replication stress, potentially contributing to genomic instability .

• Enhanced proliferative capacity: Functional experiments demonstrate that CKS2 knockdown significantly decreases cancer cell proliferation and invasion while promoting apoptosis, indicating its direct role in maintaining malignant phenotypes .

• Immune evasion: CKS2 expression shows negative correlation with immune cell infiltration in tumors, suggesting a role in modulating the tumor immune microenvironment and potentially contributing to immune escape mechanisms .

These mechanisms collectively explain how CKS2 overexpression contributes to aggressive cancer phenotypes and poor clinical outcomes across multiple tumor types .

What is the relationship between CKS2 expression and tumor immune microenvironment?

Single-sample Gene Set Enrichment Analysis (ssGSEA) and TIMER database analysis demonstrate a significant negative correlation between CKS2 expression and immune cell infiltration in lung adenocarcinoma. Specifically:

• Tumors with high CKS2 expression show reduced infiltration of:

  • CD8+ T cells

  • Dendritic cells

  • B cells

  • Natural killer cells

• This negative association with immune infiltrates suggests CKS2 may influence tumor immune evasion mechanisms and potentially impact immunotherapy response.

• The correlation with PD-L1 expression further supports CKS2's involvement in immune checkpoint regulation, though the precise mechanistic relationship requires further investigation.

These findings indicate that CKS2 expression may serve as a potential biomarker for predicting immunotherapy response and offer insights into combined therapeutic strategies targeting both CKS2 and immune pathways .

What are the recommended protocols for CKS2 detection by Western blotting?

For optimal Western blot detection of CKS2:

• Sample preparation:

  • Extract protein from cells using standard lysis buffers containing protease inhibitors

  • Load 10-20 μg of protein per lane (based on validated protocols)

• Gel electrophoresis and transfer:

  • Use higher percentage gels (12-15%) due to CKS2's low molecular weight (10 kDa)

  • Transfer to PVDF membrane (0.2 μm pore size recommended for small proteins)

• Antibody incubation:

  • Block with 5% non-fat milk or BSA

  • Incubate with primary antibody at 1/1000 dilution (e.g., Abcam's EPR7946(2) clone)

  • Validated in multiple cell lines including Caco-2, 293T, and MOLT4

• Detection and visualization:

  • Use HRP-conjugated secondary antibodies and enhanced chemiluminescence

  • Expected band size: 10 kDa

• Controls and validation:

  • Include positive controls (cell lines with known CKS2 expression)

  • Consider using CKS2 knockdown or overexpression samples for antibody validation

This protocol has been validated for detection of endogenous CKS2 across multiple human cell lines and provides reliable results for quantitative expression analysis .

How can researchers effectively use CKS2 antibodies for immunofluorescence studies?

For successful immunofluorescence detection of CKS2:

• Cell preparation:

  • Culture cells on coverslips or appropriate imaging chambers

  • Fix with 4% paraformaldehyde (PFA) for 15 minutes at room temperature

  • Permeabilize with 0.1% Triton X-100 for 10 minutes

• Antibody selection and staining:

  • Use validated antibodies for ICC/IF applications (e.g., Abcam's rabbit polyclonal)

  • Apply primary antibody at 4 μg/ml concentration

  • Incubate overnight at 4°C or 1-2 hours at room temperature

  • Use fluorophore-conjugated secondary antibodies appropriate for your microscopy setup

• Counterstaining and mounting:

  • Counterstain nuclei with DAPI

  • Mount using anti-fade mounting medium

• Imaging considerations:

  • CKS2 typically shows both nuclear and cytoplasmic localization

  • Use confocal microscopy for precise subcellular localization studies

  • Consider co-staining with cell cycle markers for functional analyses

• Controls:

  • Include secondary-only controls

  • Consider siRNA knockdown controls for specificity validation

This protocol has been successfully implemented for CKS2 detection in A549 cells and can be adapted for other cell types based on specific research requirements .

What approaches can be used to study the functional role of CKS2 in cancer cells?

Multiple complementary approaches can be employed to investigate CKS2 function in cancer:

• Gene expression modulation:

  • siRNA/shRNA-mediated knockdown to assess loss-of-function effects

  • CRISPR-Cas9 genome editing for complete knockout

  • Overexpression systems to evaluate gain-of-function effects

• Functional assays:

  • Proliferation assays (MTT, BrdU incorporation, colony formation)

  • Migration and invasion assays (transwell, wound healing)

  • Apoptosis detection (Annexin V/PI staining, caspase activity)

  • Cell cycle analysis (flow cytometry with PI staining)

• Molecular pathway analysis:

  • Co-immunoprecipitation to identify CKS2 protein interactions

  • ChIP-seq to determine transcriptional targets

  • RNA-seq for global transcriptional impact after CKS2 modulation

• In vivo models:

  • Xenograft models using CKS2-modulated cancer cells

  • Patient-derived xenografts with varying CKS2 expression levels

  • Genetically engineered mouse models for tissue-specific alterations

• Clinical correlation:

  • IHC analysis of patient samples with survival correlation

  • Integration with multi-omics patient data

These approaches have successfully demonstrated that CKS2 knockdown decreases invasion and proliferation of lung adenocarcinoma cells while promoting apoptosis, confirming its role in maintaining malignant phenotypes .

How can researchers integrate CKS2 expression data with other molecular features for comprehensive cancer analysis?

Advanced multi-omics integration approaches for CKS2 research include:

• Correlation with genomic alterations:

  • Analyze associations between CKS2 expression and specific mutations (e.g., TP53 status)

  • Evaluate copy number variations affecting CKS2 locus

  • Assess for potential synthetic lethal interactions

• Epigenetic regulation analysis:

  • Examine DNA methylation status of CKS2 promoter regions

  • Research indicates CKS2 overexpression correlates with DNA hypomethylation

  • Integrate histone modification data to understand chromatin-level regulation

• Transcriptomic integration:

  • Perform co-expression network analysis to identify functionally related genes

  • Apply differential gene expression analysis between high and low CKS2 expression groups

  • Implement pathway enrichment methods (GO, KEGG, GSEA) to identify associated biological processes

• Proteomics and interactome mapping:

  • Identify protein-protein interactions through mass spectrometry

  • Focus on cyclin-dependent kinase interactions and regulatory networks

  • Map post-translational modifications affecting CKS2 function

• Clinical data integration:

  • Correlate with treatment response data across therapeutic modalities

  • Integrate with immune infiltration metrics for immunotherapy predictions

  • Develop and validate prognostic signatures incorporating CKS2

This integrative approach has successfully revealed that CKS2 overexpression correlates with advanced stage, TP53 status, PD-L1 expression, and DNA hypomethylation in lung adenocarcinoma, providing a comprehensive molecular portrait of CKS2's role in cancer biology .

What are the implications of CKS2's role in cell cycle checkpoint override for cancer therapy?

CKS2's ability to override the intra-S-phase checkpoint has significant therapeutic implications:

• Synthetic lethality approaches:

  • Cells with CKS2 overexpression may be more vulnerable to agents targeting DNA repair mechanisms

  • PARP inhibitors or ATR inhibitors might show enhanced efficacy in CKS2-high tumors

  • Combination with conventional DNA-damaging agents may increase therapeutic window

• Cell cycle-targeted therapies:

  • CDK inhibitors may show differential efficacy based on CKS2 expression

  • Checkpoint kinase inhibitors (Chk1/2) could sensitize CKS2-overexpressing cells

  • WEE1 inhibitors might counteract the checkpoint override effect

• Resistance mechanism considerations:

  • CKS2 overexpression may contribute to chemotherapy resistance by allowing continued replication despite DNA damage

  • Monitoring CKS2 expression changes during treatment may predict acquired resistance

  • Targeting CKS2 directly could potentially re-sensitize resistant tumors

• Biomarker applications:

  • CKS2 expression might predict response to S-phase targeting agents

  • Rational combinations could be designed based on CKS2 status

  • Patient stratification for clinical trials should consider CKS2 expression

The mechanistic findings that CKS2 binding to CDK2 confers partial resistance to inhibitory tyrosine phosphorylation, allowing continued DNA replication under replicative stress, provides a biological foundation for these therapeutic strategies .

How can researchers interrogate the relationship between CKS2 and immune infiltration in tumor samples?

To investigate CKS2-immune cell relationships in tumors:

• Computational methods:

  • Single-sample Gene Set Enrichment Analysis (ssGSEA) to quantify immune cell populations

  • TIMER database analysis for estimation of immune infiltration

  • Correlation analysis between CKS2 expression and immune cell signature genes

  • Deconvolution algorithms (e.g., CIBERSORT, MCP-counter) for immune cell profiling

• Experimental validation approaches:

  • Multiplex immunohistochemistry to co-localize CKS2 and immune markers

  • Flow cytometry analysis of tumor-infiltrating lymphocytes in relation to CKS2 expression

  • Single-cell RNA sequencing to characterize immune populations in CKS2-high vs. low regions

  • Spatial transcriptomics for geographical relationships between CKS2+ cells and immune cells

• Functional interrogation:

  • Co-culture experiments with immune cells and CKS2-modulated cancer cells

  • Cytokine profiling in CKS2-high vs. low conditions

  • In vivo models with immune competent mice to assess immunotherapy response

• Clinical correlation:

  • Analysis of immunotherapy response data stratified by CKS2 expression

  • Integration with PD-L1 expression and other immune checkpoint molecules

  • Evaluation of neoadjuvant immunotherapy specimens for changes in CKS2-immune relationships

These approaches can help elucidate the mechanistic basis for the observed negative correlation between CKS2 expression and immune cell infiltration, potentially informing immunotherapeutic strategies .

What are the common challenges in CKS2 detection and quantification?

Researchers frequently encounter several challenges when working with CKS2:

• Protein size limitations:

  • CKS2's small size (10 kDa) makes it challenging to detect by Western blot

  • Recommended solutions: Use higher percentage gels (15-20%), smaller pore size membranes (0.2 μm), and optimized transfer conditions for small proteins

  • Consider using specialized gel systems designed for low molecular weight proteins

• Antibody specificity issues:

  • Potential cross-reactivity with CKS1 due to sequence homology

  • Validation strategies: Use recombinant protein controls, CKS2 knockout/knockdown samples, and peptide competition assays

  • Verify specificity across multiple applications and sample types

• Quantification challenges:

  • Dynamic range limitations in immunohistochemistry scoring

  • Standardization approach: Use automated image analysis, implement consistent scoring criteria, and include reference standards

  • Consider multiplexed approaches for contextual assessment

• Sample preparation variables:

  • Fixation artifacts in tissue samples affecting epitope accessibility

  • Solutions: Optimize antigen retrieval methods, test multiple antibody clones, and validate with fresh frozen samples when possible

• Expression level variations:

  • Cell cycle-dependent expression patterns complicating interpretation

  • Approach: Synchronize cells for in vitro studies and correlate with cell cycle markers in tissue analyses

Addressing these challenges through methodological optimization is essential for generating reliable and reproducible CKS2 research data .

How should researchers interpret contradictory findings regarding CKS2 expression across different cancer studies?

When encountering discrepancies in CKS2 research:

• Methodological differences assessment:

  • Compare detection methods (IHC vs. Western blot vs. RT-PCR)

  • Evaluate antibody clones and validation status

  • Consider scoring systems and quantification approaches

  • Assess sample processing variations (fixation time, antigen retrieval)

• Biological context considerations:

  • Tumor heterogeneity may explain sampling variations

  • Cell type-specific effects might drive apparent contradictions

  • Molecular subtype differences could contribute to variable findings

  • Disease stage variations often account for expression discrepancies

• Technical validation approaches:

  • Validate key findings using orthogonal methods

  • Confirm with multiple antibodies targeting different epitopes

  • Correlate protein with mRNA expression

  • Use genetic manipulation to confirm functional observations

• Data integration strategies:

  • Meta-analysis approaches to identify consistent patterns

  • Consider larger datasets with adequate statistical power

  • Account for covariates that might explain differences

  • Stratify analyses by relevant molecular or clinical parameters

• Reporting considerations:

  • Clearly document methodological details

  • Specify antibody validation criteria

  • Report negative and contradictory findings

  • Discuss limitations transparently

These approaches help reconcile apparently contradictory findings and contribute to a more nuanced understanding of CKS2's context-dependent roles in cancer biology .

What controls are essential for validating CKS2 antibody specificity in research applications?

Comprehensive validation of CKS2 antibodies requires multiple controls:

• Positive and negative tissue/cell controls:

  • Known CKS2-expressing cells (e.g., proliferating cancer cell lines)

  • Low-expressing normal tissues for comparison

  • Cell cycle-synchronized populations (CKS2 expression varies with cell cycle)

• Genetic manipulation controls:

  • siRNA/shRNA knockdown samples

  • CRISPR/Cas9 knockout cells

  • Overexpression systems with tagged constructs

• Peptide competition assays:

  • Pre-incubation of antibody with immunizing peptide

  • Demonstration of signal reduction with increasing peptide concentration

  • Specificity confirmation with non-target peptides

• Multiple antibody validation:

  • Comparison of different clones targeting distinct epitopes

  • Cross-validation between monoclonal and polyclonal antibodies

  • Verification across different applications (WB, IHC, IF)

• Recombinant protein standards:

  • Purified CKS2 protein as positive control

  • Related family members (e.g., CKS1) to assess cross-reactivity

  • Concentration curves to determine detection limits