CUL2 Antibody

What is CUL2 and what is its biological significance?

CUL2 (Cullin 2) is a scaffold protein for Ring finger type E3 ligases that participates in proteolysis through the ubiquitin-proteasome pathway. As one of seven human cullin proteins (CUL1-3, CUL4A, CUL4B, CUL5, and CUL7), it plays critical roles in protein degradation mechanisms. The protein has a calculated and observed molecular weight of 87 kDa. CUL2 is predominantly localized in the nucleus of various cell types, including A-431, U-251MG, and PC-3 cells, as confirmed by immunofluorescence studies . Importantly, CUL2 has been implicated in the development and progression of multiple cancer types, making it a significant protein for oncology research .

What types of CUL2 antibodies are available for research applications?

Several types of CUL2 antibodies are available for research applications:

Recombinant rabbit monoclonal antibodies offer advantages including better specificity and sensitivity, lot-to-lot consistency, and are animal origin-free, which may be important considerations for certain research applications .

How can I validate the specificity of a CUL2 antibody?

Validating CUL2 antibody specificity is crucial for reliable research. Consider implementing these methodological approaches:

• siRNA knockdown validation: Transfect cells (e.g., NTERA-2 cl.D1) with CUL2-specific siRNAs and compare Western blot results with non-targeting scrambled siRNA and untransfected controls. A significant reduction in signal intensity in the knockdown cells confirms antibody specificity .

• Peptide competition assay: Pre-incubate the antibody with an immunogenic peptide before application. The disappearance or significant reduction of signal confirms specificity, as demonstrated in immunofluorescence and flow cytometry experiments .

• Multiple cell line testing: Verify consistent detection of the expected 87 kDa band across different cell lines such as HEK-293, PC-3, HeLa, and HepG2 .

• Cross-reference with orthogonal methods: Compare results across multiple detection methods (WB, IHC, IF) to confirm target recognition consistency.

What are the optimal protocols for using CUL2 antibodies in Western blotting?

For optimal Western blotting results with CUL2 antibodies, follow these methodological guidelines:

• Sample preparation: Extract whole cell lysates from your cells of interest (successfully tested with HEK-293, PC-3, HeLa, and HepG2 cells) .

• Gel electrophoresis: Use 10% Bis-Tris gels for optimal separation of the 87 kDa CUL2 protein .

• Dilution optimization:

  • For polyclonal antibody 10981-2-AP: Use at 1:1000-1:6000 dilution

  • For recombinant monoclonal antibody 700179: Use at approximately 1 μg/mL

• Secondary antibody: Apply HRP-conjugated anti-rabbit IgG secondary antibody (e.g., 1:2500 to 1:4000 dilution) .

• Detection: Use ECL Western blotting substrate for chemiluminescent detection .

• Expected result: A clear band at approximately 87-89 kDa corresponding to CUL2 .

This protocol has been validated to produce consistent results across multiple cell lines and can be adapted based on your specific experimental conditions.

How should I optimize immunohistochemistry protocols for CUL2 detection in tissue samples?

For effective immunohistochemical detection of CUL2 in tissue samples:

• Tissue preparation: Use formalin-fixed, paraffin-embedded (FFPE) tissue sections. Various tissue types have been successfully tested, including human prostate cancer tissue, lung, and ovarian carcinoma .

• Antigen retrieval: Two effective methods have been validated:

  • Primary recommendation: TE buffer at pH 9.0

  • Alternative method: Citrate buffer at pH 6.0

  • For certain tissues like lung and ovarian carcinoma, EDTA pretreatment has proven effective

• Antibody dilution:

  • For polyclonal antibody (10981-2-AP): Use 1:50-1:500 dilution

  • For monoclonal antibody (700179): Apply at 5 μg/mL

• Visualization: Implement DAB (3,3'-diaminobenzidine) staining for detection .

• Magnification: Examine results at 20× magnification for optimal visualization of cellular localization patterns .

This optimized protocol allows for specific detection of CUL2 in cancer and normal tissues, enabling comparative analysis of expression levels.

What are the best practices for immunofluorescence detection of CUL2?

For successful immunofluorescence detection of CUL2:

• Cell preparation: HeLa cells have been validated for clear CUL2 visualization .

• Antibody application: For recombinant rabbit monoclonal antibody (700179), use at 10 μg/mL dilution .

• Secondary detection: Apply Alexa Fluor 488-conjugated goat anti-rabbit secondary antibody at 1:1000 dilution .

• Counterstaining options:

  • Nuclear staining: Hoechst dye

  • Cytoskeletal visualization: Alexa Fluor 568 Phalloidin

• Controls: Include a peptide competition assay by pre-incubating the antibody with the immunogenic peptide to confirm specificity .

The expected result is predominantly nuclear localization of CUL2, consistent with its reported subcellular distribution in multiple cell lines .

How can CUL2 antibodies be utilized for studying protein-protein interactions?

CUL2 antibodies can effectively investigate protein-protein interactions through these methodological approaches:

• Co-immunoprecipitation (CoIP): CUL2 polyclonal antibody (10981-2-AP) has been validated for CoIP applications, allowing the identification of CUL2 binding partners . The procedure involves:

  • Using 0.5-4.0 μg of antibody for 1.0-3.0 mg of total protein lysate

  • Precipitating the protein complex with appropriate beads

  • Analyzing the precipitated proteins by Western blotting to identify interacting partners

• Network analysis approach: Computational prediction tools like GeneMANIA can supplement experimental findings by identifying potential biological associations of CUL2. This approach has revealed functional partners linked to integumentary, genetic, immune, endocrine, urinary, and gastrointestinal systems .

• PPI network construction: Creating protein-protein interaction networks based on experimental CoIP data and database mining can reveal novel functional associations for CUL2 and guide further experimental validation .

By combining these experimental and computational approaches, researchers can comprehensively map the CUL2 interactome and understand its role in various cellular processes.

What methodologies can be used to investigate CUL2's role in cancer development and progression?

To investigate CUL2's role in cancer, implement these research methodologies:

• Expression profiling across cancer types: Analyze CUL2 expression patterns using databases like TCGA and GTEx. Current evidence shows CUL2 is significantly upregulated in multiple cancer types including ACC, BLCA, BRCA, CESC, CHOL, COAD, ESCA, GBM, HNSC, LGG, LIHC, LUAD, LUSC, OV, PAAD, PRAD, SKCM, STAD, THCA, and UCS, while downregulated in LAML compared to normal tissues .

• Protein expression validation:

  • Western blotting and qRT-PCR on clinical tissue samples have confirmed elevated CUL2 levels in hepatocellular carcinoma compared to adjacent normal tissues

  • Immunohistochemistry using CUL2 antibodies at 1:50-1:500 dilution on cancer tissues such as prostate cancer

• Functional studies via knockdown/knockout:

  • Use siRNA-mediated knockdown of CUL2 (validated in multiple publications) to assess effects on cancer cell phenotypes

  • Evaluate changes in proliferation, migration, invasion, and apoptosis

• Tumor microenvironment analysis: Employ the TIMER database to examine correlations between CUL2 expression and immune cell infiltration in different cancer types, offering insights into potential immunotherapeutic strategies .

• Survival analysis: Conduct Kaplan-Meier analysis to investigate associations between CUL2 expression levels and patient prognosis across different cancer types .

These combined approaches provide a comprehensive understanding of CUL2's oncogenic roles and potential as a diagnostic or therapeutic target.

How can flow cytometry be optimized for CUL2 detection in cancer research?

For optimal flow cytometric detection of CUL2 in cancer research:

• Cell preparation: Use Jurkat cells or other cancer cell lines of interest. Fix and permeabilize cells using appropriate reagents like FIX & PERM .

• Antibody concentration: Apply CUL2 recombinant rabbit monoclonal antibody (700179) at 0.5 μg per sample .

• Secondary detection: Implement Alexa Fluor 488 goat anti-rabbit IgG for fluorescent detection .

• Controls:

  • Negative control: Omit primary antibody

  • Specificity control: Pre-incubate with immunogenic peptide (should show decreased signal)

• Analysis parameters: Gate for live, single cells before analyzing CUL2 expression.

• Applications: This method is particularly valuable for:

  • Quantifying CUL2 expression levels across different cancer cell populations

  • Correlating CUL2 expression with other cancer markers

  • Sorting CUL2-high versus CUL2-low cell populations for functional studies

This protocol enables precise quantification of CUL2 expression across cell populations and facilitates investigation of its heterogeneity in cancer samples.

What are common challenges with CUL2 antibodies and how can they be addressed?

Researchers commonly encounter these challenges when working with CUL2 antibodies:

• Background signals in Western blotting:

  • Solution: Optimize blocking conditions (try 5% skimmed milk)

  • Solution: Increase washing steps and duration

  • Solution: Titrate antibody concentration (start with 1:1000 dilution for polyclonal and 1 μg/mL for monoclonal antibodies)

• Weak or absent signals in IHC:

  • Solution: Optimize antigen retrieval method (compare TE buffer pH 9.0 vs. citrate buffer pH 6.0)

  • Solution: Increase antibody concentration (try 1:50 dilution for polyclonal antibodies)

  • Solution: Extend primary antibody incubation time (overnight at 4°C)

• Non-specific binding in immunofluorescence:

  • Solution: Include a peptide competition control to confirm specificity

  • Solution: Optimize fixation and permeabilization conditions

  • Solution: Consider using recombinant monoclonal antibodies for improved specificity

• Inconsistent results between experiments:

  • Solution: Aliquot antibody upon receipt to avoid freeze-thaw cycles

  • Solution: Store according to manufacturer recommendations (-20°C, avoid repeated freeze-thaw)

  • Solution: Standardize lysate preparation methods

By implementing these troubleshooting strategies, researchers can significantly improve the reliability and reproducibility of their CUL2 antibody-based experiments.

How should CUL2 antibodies be stored and handled to maintain optimal activity?

For optimal maintenance of CUL2 antibody activity:

• Storage temperature:

  • Store unopened antibody at -20°C

  • Avoid storage at 4°C for extended periods unless explicitly stated by manufacturer

• Aliquoting strategy:

  • Upon receipt, divide into small aliquots to prevent repeated freeze-thaw cycles

  • For polyclonal antibody 10981-2-AP, aliquoting is unnecessary for -20°C storage

  • For A02986 antibody, aliquoting is recommended before freezing

• Buffer composition:

  • Most CUL2 antibodies are provided in PBS with 0.02% sodium azide and 50% glycerol at pH 7.3

  • Some may contain 0.1% BSA in smaller (20μL) sizes

• Thawing procedure:

  • Thaw antibodies on ice or at 4°C

  • Centrifuge product if not completely clear after standing at room temperature

• Dilution recommendations:

  • Dilute only prior to immediate use

  • Do not store diluted antibody for extended periods

• Expiration considerations:

  • Antibodies are typically stable for one year from date of opening when properly stored

  • For shipping, cold packs or dry ice are recommended

Following these storage and handling recommendations will ensure consistent antibody performance across experiments and maximize shelf life.

How does CUL2 expression correlate with cancer prognosis and patient outcomes?

Comprehensive analysis of CUL2 expression and its correlation with cancer prognosis reveals:

• Expression patterns: CUL2 is significantly upregulated in numerous cancer types including ACC, BLCA, BRCA, CESC, CHOL, COAD, ESCA, GBM, HNSC, LGG, LIHC, LUAD, LUSC, OV, PAAD, PRAD, SKCM, STAD, THCA, and UCS, while downregulated in LAML compared to normal tissues .

• Protein validation: Immunohistochemistry and Western blot analyses confirm increased CUL2 protein expression in cancer tissues, particularly in hepatocellular carcinoma compared to adjacent non-tumor tissues .

• Survival associations: Kaplan-Meier analysis has demonstrated significant correlations between CUL2 expression levels and patient survival outcomes across different cancer types .

• Mechanistic implications: As a scaffold protein for Ring finger type E3 ligases involved in proteolysis through the ubiquitin-proteasome pathway, CUL2 likely influences cancer progression by regulating the degradation of tumor suppressors or stability of oncoproteins .

• Immunotherapy relevance: Analysis of the tumor microenvironment has revealed associations between CUL2 expression and immune cell infiltration patterns, suggesting potential implications for immunotherapy efficacy .

These findings collectively suggest that CUL2 may serve as a valuable prognostic marker in multiple cancer types, with potential applications in predicting treatment responses, particularly to immunotherapies.

What are the current understanding and methodologies for studying CUL2's role in immune cell infiltration?

Current research methodologies for investigating CUL2's relationship with immune cell infiltration include:

• Correlation analysis using TIMER database:

  • Spearman correlation analysis has been employed to generate heatmaps comparing CUL2 mRNA expression with infiltration levels of different immune cell subtypes

  • This approach provides insight into how CUL2 may influence the tumor immune microenvironment

• Tumor microenvironment (TME) assessment:

  • Focused analysis of non-tumor constituents within the tumor microenvironment

  • Examination of how CUL2 expression correlates with various immune cell populations and their functional states

• Integration of multi-omics data:

  • Combining CUL2 expression data with immune cell signatures and clinical outcomes

  • Analysis of correlations between CUL2 and expression of immunomodulators

• Immunotherapy cohort studies:

  • Investigation of associations between CUL2 expression and response to immunotherapy

  • Potential for developing CUL2 as a predictive biomarker for immunotherapy efficacy

These methodological approaches collectively contribute to understanding how CUL2 may influence the immune landscape within tumors, potentially guiding the development of novel immunotherapeutic strategies across different cancer types.

How can gene set enrichment analysis (GSEA) be applied to understand CUL2's functional impact in cancer?

Gene Set Enrichment Analysis (GSEA) offers powerful methodological approaches for investigating CUL2's functional impact in cancer:

• Implementation methodology:

  • Group patient samples based on CUL2 expression levels (high vs. low)

  • Apply GSEA computational methods to identify significantly enriched gene sets associated with CUL2 expression

  • Analyze both positively and negatively correlated pathways

• Biological pathway identification:

  • GSEA reveals key biological processes and signaling pathways affected by CUL2 expression

  • Identifies molecular mechanisms through which CUL2 may influence cancer progression

• Integration with functional data:

  • Correlate GSEA findings with experimental data from CUL2 knockdown/overexpression studies

  • Validate predicted pathways through targeted molecular assays

• Cross-cancer type comparison:

  • Apply GSEA across multiple cancer types to identify common and distinct CUL2-associated pathways

  • Determine cancer-specific versus universal functions of CUL2

• Therapeutic implications:

  • Identify potential drug targets within CUL2-enriched pathways

  • Predict synergistic therapeutic combinations based on pathway analysis

This comprehensive GSEA approach provides mechanistic insights into how CUL2 influences cancer biology beyond simple expression correlations, guiding more targeted experimental designs and potentially revealing novel therapeutic strategies .