CCNB2 Antibody

What is CCNB2 and what is its biological function?

CCNB2 (Cyclin B2) belongs to the B-type cyclin family and functions as a critical cell cycle regulator. Located on chromosome 15q22, it binds to cyclin-dependent kinases (CDKs) to regulate their activities . CCNB2 is synthesized during the G1 phase in cancer cells and downregulated at anaphase . It primarily participates in G2/M phase transformation in the eukaryotic cell cycle by activating CDKs . Defects in CCNB2 function can lead to abnormal cell cycle progression and contribute to tumorigenesis . The protein is predominantly localized in the cytoplasm of cells .

What types of CCNB2 antibodies are available for research applications?

Several types of CCNB2 antibodies are available for research, categorized by:

• Target specificity:

  • Total CCNB2 antibodies that recognize the protein regardless of post-translational modifications

  • Phospho-specific antibodies like Anti-Phospho-CCNB2(S22) that target specific phosphorylated residues

• Host species:

  • Rabbit-derived antibodies (most common)

• Clonality:

  • Polyclonal antibodies for broader epitope recognition

  • Monoclonal antibodies for specific epitope targeting

• Applications:

  • Antibodies optimized for immunohistochemistry (IHC)

  • Antibodies validated for immunoblotting/Western blotting

  • Antibodies for dot blotting applications

What are the recommended storage conditions for CCNB2 antibodies?

For optimal preservation of antibody activity, CCNB2 antibodies should be stored according to manufacturer specifications. Generally:

• Short-term storage: 2-8°C (refrigerated)

• Long-term storage: -20°C (frozen)

• Avoid repeated freeze-thaw cycles to prevent protein denaturation

• Typical shelf life: 12 months from the date of shipment when stored properly

For some applications, small aliquots may be prepared to minimize freeze-thaw cycles. Always refer to the specific antibody's datasheet for optimal storage conditions, as they may vary between products and manufacturers.

What are the recommended dilutions and protocols for CCNB2 antibodies in different applications?

IHC Protocol Outline:

• Deparaffinize and rehydrate tissue sections

• Perform antigen retrieval (method may vary based on specific antibody)

• Block endogenous peroxidase activity

• Apply primary anti-CCNB2 antibody at 1:500 dilution

• Incubate at optimal temperature (typically 4°C overnight)

• Apply appropriate secondary antibody

• Develop with chromogen

• Counterstain, dehydrate, and mount

Scoring System for IHC:

• Cytoplasmic staining intensity: 0 = negative, 1 = weak, 2 = moderate, 3 = strong

• Percentage of staining (0-100%)

• Calculate H-score = intensity × percentage (range 0-300)

How should CCNB2 antibody specificity be validated for research applications?

Thorough validation is essential to ensure reliable research results:

• Positive and negative controls:

  • Use tissues known to express CCNB2 (e.g., TNBC tissues) as positive controls

  • Use normal breast tissues as negative controls or tissues with minimal expression

• Genetic knockdown validation:

  • Validate specificity using CCNB2 knockdown cells (shRNA or siRNA)

  • Compare expression in control vs. CCNB2-depleted samples by immunoblotting

• Phospho-antibody validation:

  • For phospho-specific antibodies, compare reactivity with phosphorylated vs. non-phosphorylated peptides

  • Perform dot blot analysis with 50 ng of phospho-peptide vs. non-phospho-peptide

• Molecular weight confirmation:

  • Verify the correct molecular weight band in Western blots

  • CCNB2 should appear at the expected size

• Subcellular localization:

  • Confirm cytoplasmic localization pattern as reported in literature

How is CCNB2 expression analyzed in cancer tissues?

CCNB2 expression in cancer tissues can be analyzed through multiple complementary approaches:

• Immunohistochemistry (IHC):

  • Primary method for protein-level detection in tissue samples

  • Scoring based on modified histochemical score (H-score)

  • Cytoplasmic staining intensity graded from 0-3

  • Percentage of positive cells estimated (0-100%)

  • H-score calculated as intensity × percentage (range 0-300)

• Transcriptomic analysis:

  • RNA sequencing or microarray data from databases like TCGA

  • Analysis of CCNB2 mRNA expression levels across cancer types

  • Comparison between tumor and normal tissues

• Quantitative PCR:

  • Forward primer: 5′-CAACCCACCAAAACAACA-3′

  • Reverse primer: 5′-AGAGCAAGGCATCAGAAA-3′

  • GAPDH as reference gene

• Western blotting:

  • Protein extraction from tissues or cell lines

  • CCNB2 antibody at 1:1000 dilution

  • β-actin as loading control (1:2000 dilution)

What is the prognostic significance of CCNB2 expression in different cancer types?

CCNB2 expression has significant prognostic value across several cancer types:

• Breast cancer:

  • High CCNB2 expression correlates with poor prognosis

  • Associated with tumor size (p = 0.022) and pTNM stage (p = 0.021) in TNBC

  • Independent predictor of poor prognosis in breast cancer

  • High expression in tumors with lymphovascular invasion (LVI)

• Low-grade glioma (LGG):

  • LGG patients with high CCNB2 expression have poorer prognosis

  • Expression correlates with WHO grade, IDH mutational status, and 1p/19q codeletion status

• Lung cancer:

  • Significantly higher expression in lung adenocarcinoma (LUAD), lung squamous cell carcinoma (LUSC), and small cell lung cancer compared to normal tissues

  • The diagnostic accuracy (SROC) values: LUAD = 0.92, LUSC = 0.97, SCLC = 0.98

• Clear cell renal cell carcinoma (ccRCC):

  • High CCNB2 expression serves as an independent predictor of poor prognosis

How does CCNB2 expression correlate with specific clinicopathological features?

CCNB2 expression correlates with several clinicopathological features that indicate more aggressive disease:

How can CCNB2 antibodies be used to study cell cycle progression?

CCNB2 antibodies provide valuable tools for investigating cell cycle regulation:

• Cell cycle phase analysis:

  • Use in combination with flow cytometry to correlate CCNB2 levels with cell cycle phases

  • Immunofluorescence detection to visualize spatiotemporal changes throughout cell cycle

  • Western blotting of synchronized cell populations to track CCNB2 expression changes

• G2/M phase transition studies:

  • Monitor CCNB2 levels as cells progress through G2 to M phase

  • Combine with CDK activity assays to correlate CCNB2 binding with kinase activation

  • Co-immunoprecipitation to identify CCNB2-interacting proteins during specific cell cycle phases

• Knockdown experiments:

  • Use CCNB2 antibodies to confirm successful knockdown in functional studies

  • Evaluate effects of CCNB2 depletion on cell cycle distribution

  • Monitor changes in downstream signaling pathways

• Multiplexed detection:

  • Combine CCNB2 antibodies with other cell cycle markers (cyclins, CDKs)

  • Use dual immunofluorescence to correlate CCNB2 with markers of specific cell cycle phases

What functional assays confirm CCNB2's role in tumor growth and proliferation?

Research has employed several functional assays to establish CCNB2's role in cancer progression:

• In vitro proliferation assays:

  • MTT assay: Demonstrated that CCNB2 knockdown significantly reduced proliferation of TNBC cells

  • Colony formation assay: Showed decreased colony formation capacity in CCNB2-depleted cancer cells

• Migration and invasion assays:

  • Wound healing/scratching assay: Revealed reduced migration rate in CCNB2 knockdown cells

  • Transwell migration assays: Demonstrated CCNB2's role in cancer cell invasiveness

  • Endothelial cell interaction assays: Showed CCNB2 knockdown reduced breast cancer cell adherence and transmigration across endothelial cells

• In vivo tumor models:

  • Xenograft models: MDA-MB-231 cells with CCNB2 knockdown showed significantly decreased tumor volume in nude mice

  • Tumor growth curves: Monitored over 29 days showed significantly smaller tumors in CCNB2-depleted groups

  • Ex vivo protein validation: Confirmed reduced CCNB2 expression in harvested tumors by immunoblotting

• Cell cycle analysis:

  • Flow cytometry demonstrated that CCNB2 knockdown blocked G2/M transition

  • Increased apoptotic cell population in CCNB2-depleted cells

How does CCNB2 expression correlate with immune infiltration in tumors?

CCNB2 expression shows significant correlations with immune cell infiltration across multiple cancer types:

• In low-grade glioma (LGG):

  • Positive correlation with B-cell infiltration (cor = 0.321, P = 5.96e-13)

  • B cells show higher relationship with LGG than other immune cell types

• In clear cell renal cell carcinoma (ccRCC):

  • Significant positive correlation with multiple immune cell types :

    • Activated CD4 T cells (ρ = 0.667, p < 0.001)

    • Activated CD8 T cells (ρ = 0.416, p < 0.001)

    • Gamma delta T cells (ρ = 0.384, p < 0.001)

    • Type 2 T helper cells (ρ = 0.377, p < 0.001)

    • T follicular helper cells (ρ = 0.349, p < 0.001)

    • Myeloid-derived suppressor cells (ρ = 0.348, p < 0.001)

• In psoriasis:

  • Negative correlation with resting mast cells, M2 macrophages, and plasma cells

  • Differential expression pattern between psoriasis and normal skin tissues

• Across multiple cancers:

  • Increased immune infiltration in renal clear cell carcinoma and thyroid cancer with elevated CCNB2

  • Increased infiltration of activated CD4 T cells and type 2 T helper cells in most tumors

What is the relationship between CCNB2 and immune checkpoint molecules?

CCNB2 expression demonstrates important connections with immune checkpoint molecules:

• Positive correlation with inhibitory immune checkpoints:

  • Significant positive correlation with multiple checkpoint molecules in ccRCC :

    • LAG-3

    • TIGIT

    • PD-1

    • PD-L1

    • CTLA-4

    • CD47

    • PTP-1B

• Potential mechanism for immune evasion:

  • High CCNB2 expression might facilitate tumor immune escape through checkpoint upregulation

  • Association with immunosuppressive cell populations including T regulatory cells and myeloid-derived suppressor cells

• Correlation with immune response in skin disorders:

  • In psoriasis, CCNB2 expression positively correlates with inflammatory markers :

    • IL17A

    • IL22

    • IL23R

    • TNF

• Implications for immunotherapy:

  • The correlation between CCNB2 and microsatellite instability and tumor mutation burden suggests CCNB2 as a candidate immunotherapy target

  • Could potentially serve as a biomarker for patient selection for checkpoint inhibitor therapy

How can phospho-specific CCNB2 antibodies advance understanding of post-translational regulation?

Phospho-specific antibodies provide unique insights into CCNB2 regulation:

• Tracking specific phosphorylation sites:

  • Anti-Phospho-CCNB2(S22) antibodies detect phosphorylation at serine 22

  • Enable monitoring of site-specific phosphorylation events during cell cycle progression

  • Allow correlation between phosphorylation status and protein function

• Kinase-substrate relationship studies:

  • Investigation of which kinases phosphorylate CCNB2 at specific residues

  • Studying the dynamics of phosphorylation during normal and malignant cell cycles

  • Correlation with CDK activity and other cell cycle regulators

• Signal transduction pathway analysis:

  • Determination of how upstream signals affect CCNB2 phosphorylation

  • Use with kinase inhibitors to map signaling pathways regulating CCNB2

  • Identification of phosphatases that regulate CCNB2 activity

• Therapeutic target identification:

  • Screening for compounds that modulate specific phosphorylation events

  • Development of inhibitors targeting kinases responsible for CCNB2 phosphorylation

  • Evaluation of phosphorylation status as biomarker for treatment response

How can CCNB2 antibodies contribute to developing therapeutic strategies?

CCNB2 antibodies can facilitate multiple approaches in therapeutic development:

• Target validation studies:

  • Confirmation of CCNB2 as a therapeutic target in specific cancer types

  • Validation of target expression in patient-derived samples

  • Correlation of expression with response to specific therapies

• Drug screening and development:

  • High-throughput screening for compounds that modulate CCNB2 expression or activity

  • Evaluation of CDK4/6 inhibitors efficacy in CCNB2-overexpressing tumors

  • Assessment of PI3K-AKT-mTOR pathway inhibitors in CCNB2-high tumors

• Biomarker development:

  • Stratification of patients based on CCNB2 expression for clinical trials

  • Development of companion diagnostics for CCNB2-targeting therapies

  • Creation of prognostic models incorporating CCNB2 status

• Combination therapy approaches:

  • Using CCNB2 antibodies to assess effectiveness of combining cell cycle inhibitors with:

    • Immune checkpoint inhibitors based on CCNB2-immune correlations

    • Conventional chemotherapies

    • Targeted therapies against pathways synergistic with CCNB2 inhibition