CDKN2C Antibody

What are the validated applications for CDKN2C antibodies?

CDKN2C antibodies have been validated for multiple research applications including:

• Western Blot (WB): Recommended dilutions range from 1:500-1:2000

• Immunohistochemistry (IHC): Recommended dilutions range from 1:50-1:200

• Immunocytochemistry/Immunofluorescence (ICC/IF): Recommended dilutions range from 1:50-1:200

• Immunoprecipitation (IP): Recommended dilution around 1:40

The applications vary slightly between antibody clones, with some monoclonal antibodies such as EPR15891 (ab192239) and SR1208 being validated for all four applications .

How should CDKN2C antibodies be stored and handled to maintain optimal activity?

For optimal antibody performance:

• Store at -20°C and avoid repeated freeze-thaw cycles

• For long-term storage: Keep at -20 to -70°C as supplied

• After reconstitution: Use within 1 month when stored at 2-8°C under sterile conditions

• For extended use after reconstitution: Store for up to 6 months at -20 to -70°C under sterile conditions

• Buffer composition typically includes PBS, pH 7.4, 150mM NaCl, and 50% glycerol

How can I confirm the specificity of my CDKN2C antibody?

To confirm antibody specificity:

• Positive controls: Use cell lines with known CDKN2C expression such as HeLa or 293T cells

• Knockout validation: Compare signal between wild-type cells and CDKN2C knockout cells (e.g., using the knockout cell line ab265031)

• Loading controls: Include appropriate loading controls such as GAPDH (anti-GAPDH antibody [6C5])

• Peptide competition: Pre-incubate the antibody with the immunizing peptide to verify signal reduction

• Multiple antibody validation: Use antibodies from different clones targeting different epitopes

Abcam's EPR15891 clone (ab192239) has undergone extensive validation showing specific reactivity with CDKN2C in wild-type HeLa cells with signal loss in knockout cells .

What are the optimal fixation and antigen retrieval methods for CDKN2C immunohistochemistry?

For optimal IHC results:

• Fixation:

  • 10% neutral buffered formalin fixation for 24-48 hours is recommended

  • Over-fixation may mask epitopes and reduce staining intensity

• Antigen retrieval:

  • Heat-induced epitope retrieval (HIER) in citrate buffer (pH 6.0) for 20 minutes

  • For difficult samples, try EDTA buffer (pH 9.0) as an alternative

• Blocking:

  • Use 5-10% normal serum from the same species as the secondary antibody

  • Include 0.1-0.3% Triton X-100 for improved penetration in tissue sections

• Incubation times:

  • Primary antibody: Overnight at 4°C at dilutions of 1:50-1:200

  • Secondary antibody: 1-2 hours at room temperature

How does CDKN2C expression correlate with patient outcomes in different cancer types?

CDKN2C expression shows variable correlation with patient outcomes across cancer types:

• Small Cell Lung Carcinoma (SCLC):

  • High CDKN2C expression is associated with poor prognosis (hazard ratio > 1, p < 0.05)

  • CDKN2C shows remarkable potential for distinguishing SCLC from non-SCLC (sensitivity, specificity, and AUC ≥ 0.95)

  • Can differentiate SCLC from NSCLC with sensitivity = 0.87, specificity = 0.94, AUC = 0.96

• Multiple Myeloma:

  • Deletion of CDKN2C at 1p32.3 occurs in 7-40% of myeloma cases

  • Homozygous deletion of CDKN2C correlates with increased proliferation

  • Cases with CDKN2C homozygous deletion were found to be among the most proliferative myelomas

• Medullary Thyroid Carcinoma (MTC):

  • CDKN2C loss has been associated with RET-mediated MTC

  • Acts as a haploinsufficient tumor suppressor gene

  • CDKN2C copy number loss occurs in approximately 38% of sporadic MTC tumors

• HBV-induced Liver Disease:

  • CDKN2C overexpression correlates with disease progression in HBV-infected patients

  • Acts as a host factor enhancing HBV replication by inducing cell cycle G1 arrest

How does CDKN2C function in immune regulation and autoimmunity?

CDKN2C plays significant roles in immune regulation:

• B cell development and autoimmunity:

  • CDKN2C deficiency promotes expansion of B1a cells, a subset implicated in autoimmunity

  • CDKN2C-deficient mice (B6.p18-/-) show significantly higher numbers of peritoneal B1a cells compared to control mice

  • B6.p18-/- mice produce significant amounts of anti-DNA IgM and IgG, indicating contribution to humoral autoimmunity

  • CDKN2C deficiency synergizes with lpr-mediated pathology in autoimmune models

• Regulation mechanism:

  • CDKN2C (p18) regulates the size of the B1a cell pool

  • The expansion of B1a cells in CDKN2C-deficient mice was normalized by cyclin D2 deficiency

  • This suggests that regulation of the G1 phase through cyclin D2 and D3/CDK complexes is critical for B1a cell proliferation

• Implications for autoimmune diseases:

  • CDKN2C may represent a potential therapeutic target for B-cell mediated autoimmune disorders

  • Modulating CDKN2C function could potentially help normalize aberrant B cell populations

What are the main factors affecting signal variability in CDKN2C detection by Western blot?

Several factors can influence CDKN2C detection via Western blot:

• Sample preparation issues:

  • CDKN2C is a nuclear protein; ensure efficient nuclear protein extraction

  • Use appropriate lysis buffers with protease inhibitors

  • Sample degradation can occur if not properly stored at -80°C

• Gel percentage considerations:

  • CDKN2C is a small protein (~18-19 kDa)

  • Use 12-15% SDS-PAGE gels for optimal resolution

  • Consider using Tricine-SDS-PAGE for better separation of small proteins

• Transfer optimization:

  • Small proteins may transfer through the membrane

  • Use PVDF membranes (0.2 μm pore size) rather than nitrocellulose

  • Reduce transfer time or voltage to prevent over-transfer

• Detection sensitivity:

  • Primary antibody concentration: Start with 1:1000 dilution and adjust as needed

  • Secondary antibody selection: Use high-sensitivity HRP or fluorescent detection systems

  • Enhanced chemiluminescence reagents may be needed for low abundance samples

• Biological variability:

  • CDKN2C expression varies across cell types and conditions

  • Expression is typically higher in cells undergoing cell cycle arrest

  • Consider cell synchronization for consistent results

How can I reconcile contradictory findings regarding CDKN2C expression in my research?

When faced with contradictory findings regarding CDKN2C expression:

• Validate using multiple detection methods:

  • Combine protein detection (Western blot, IHC) with mRNA analysis (qRT-PCR)

  • Employ different antibody clones that target different epitopes of CDKN2C

  • Use functional assays to assess CDKN2C activity not just expression levels

• Consider post-translational modifications:

  • CDKN2C function can be regulated by phosphorylation and other modifications

  • These may affect antibody recognition but not necessarily function

• Tissue/cell heterogeneity:

  • CDKN2C expression varies across cell types within heterogeneous samples

  • In adipose tissue, CDKN2C protein was found mainly expressed in adipocytes compared to stromal vascular cells

  • Consider single-cell analysis or cell sorting to resolve heterogeneity

• Context-dependent regulation:

  • CDKN2C expression in subcutaneous adipose tissue (SAT) is inversely correlated with measures of hyperglycemia, insulin resistance, and visceral adiposity

  • Expression can be upregulated during certain differentiation processes (e.g., adipocyte differentiation)

  • Consider the specific biological context and disease state of your samples

What are the most effective methods for CDKN2C knockdown or knockout in experimental models?

Multiple effective strategies exist for CDKN2C genetic modification:

• CRISPR/Cas9 gene editing:

  • Successfully used for CDKN2C knockdown in human preadipocytes

  • Target design should focus on early exons to ensure complete functional disruption

  • Verification via Western blot is essential as CRISPR can produce in-frame mutations

• siRNA/shRNA approaches:

  • Transient knockdown via siRNA transfection (typically 48-72 hours)

  • Stable knockdown using lentiviral shRNA delivery

  • Multiple target sequences should be tested to identify optimal knockdown efficiency

• Knockout mice models:

  • B6.p18-/- mice have been established and show expanded B1a cell populations

  • These models can be crossed with other genetically modified mice to study interactions (e.g., B6.p18-/-.lpr mice)

• Verification methods:

  • Confirm knockdown/knockout at both mRNA level (qRT-PCR) and protein level (Western blot)

  • Functional assays such as cell cycle analysis should be included

  • For partial knockdowns, quantify the degree of reduction accurately

How do CDKN2C knockout phenotypes vary across different cell types and tissues?

CDKN2C knockout produces diverse phenotypes depending on cell type and tissue context:

How does CDKN2C interact with the immune microenvironment in cancer?

CDKN2C has significant interactions with the immune microenvironment in cancer:

• Immune infiltration correlation:

  • CDKN2C expression correlates with immune infiltration in various cancer types

  • Relationship between CDKN2C expression and immune microenvironment has been examined using both TIMER and ESTIMATE algorithms

  • TIMER scores evaluate infiltration levels of six immune cell types: B cells, CD4 T cells, CD8 T cells, neutrophils, macrophages, and dendritic cells

• Stromal and immune scores:

  • ESTIMATE scores (stromal, immune, and composite ESTIMATE scores) show correlation with CDKN2C expression

  • These correlations provide insights into how CDKN2C may influence the tumor microenvironment

• Immunotherapy implications:

  • CDKN2C expression is related to immune checkpoint molecules

  • Investigation using TISIDB has revealed associations between CDKN2C expression and immune-related genes

  • These include major histocompatibility complex molecules, immunoinhibitory genes, and immunostimulatory genes

  • Suggests potential usefulness of CDKN2C as a prognostic marker in immunotherapy

What approaches can resolve inconsistencies in CDKN2C antibody staining patterns across different tissue samples?

To resolve inconsistent CDKN2C antibody staining patterns:

• Optimized antigen retrieval protocols:

  • Test multiple antigen retrieval methods (citrate buffer pH 6.0, EDTA buffer pH 9.0, enzymatic retrieval)

  • Optimize duration and temperature of antigen retrieval

  • For difficult tissues, combined approaches may be necessary

• Antibody validation strategy:

  • Use multiple antibodies targeting different epitopes of CDKN2C

  • Include positive controls (cells/tissues with known high CDKN2C expression)

  • Include negative controls (CDKN2C knockout tissues or blocking peptides)

• Signal amplification techniques:

  • For low expression samples, consider tyramide signal amplification

  • Polymer-based detection systems can improve sensitivity

  • Fluorescent multiplexing can help distinguish true signal from background

• Tissue-specific considerations:

  • Modify fixation protocols based on tissue type

  • Account for endogenous biotin in liver and kidney tissues

  • Consider tissue-specific autofluorescence quenching for IF applications

• Quantitative analysis:

  • Implement digital pathology scoring systems

  • Use staining intensity scoring criteria as described in published studies

  • For example, the study examining CDKN2C in SCLC used standardized protein staining scoring criteria

How might CDKN2C function as a biomarker or therapeutic target in metabolic diseases?

Emerging evidence suggests CDKN2C's potential role in metabolic diseases:

• Adipose tissue metabolism:

  • CDKN2C mRNA expression in subcutaneous and omental adipose tissue is reduced in Type 2 Diabetes (T2D) and obese subjects

  • Expression inversely correlates with measures of hyperglycemia, insulin resistance, and visceral adiposity

  • Positively correlates with expression of genes in metabolic pathways including insulin signaling, fatty acid and carbohydrate metabolism

• Adipocyte differentiation:

  • CDKN2C protein expression is upregulated during adipocyte differentiation

  • Knockdown of CDKN2C reduces expression of adipocyte differentiation markers (CEBPA, ADIPOQ, FASN)

  • Suggests a role in adipogenesis and lipid storage capacity

• Potential therapeutic implications:

  • Targeting CDKN2C expression might improve adipose tissue function in insulin resistance

  • Could enhance subcutaneous adipose tissue lipid storage capacity

  • Might reduce ectopic fat accumulation associated with metabolic disease

• Biomarker potential:

  • CDKN2C expression levels in adipose tissue may serve as a biomarker for insulin sensitivity

  • Could potentially identify patients at risk for metabolic complications

  • Longitudinal studies would be needed to validate prognostic value

What are the latest technical advances in detecting low-abundance CDKN2C in clinical samples?

Recent technical advances for detecting low-abundance CDKN2C include:

• High-sensitivity digital PCR:

  • Allows absolute quantification of CDKN2C copy number and expression

  • Can detect single-copy loss in heterogeneous samples

  • Essential for identifying CDKN2C haploinsufficiency in cancer samples

• Advanced immunohistochemistry approaches:

  • Tyramide signal amplification (TSA) can increase detection sensitivity 10-100 fold

  • Multiplex immunofluorescence allows simultaneous detection of CDKN2C with other markers

  • Automated quantitative analysis (AQUA) for objective quantification of protein levels

• Mass spectrometry-based proteomics:

  • Targeted proteomics using selected reaction monitoring (SRM) or parallel reaction monitoring (PRM)

  • Enables absolute quantification of CDKN2C protein without antibody dependency

  • Can detect post-translational modifications affecting protein function

• Liquid biopsy approaches:

  • Detection of circulating tumor DNA with CDKN2C alterations

  • Analysis of CDKN2C methylation patterns in cell-free DNA

  • Potential for non-invasive monitoring of diseases with CDKN2C involvement

• Single-cell technologies:

  • Single-cell RNA sequencing for heterogeneity analysis

  • Mass cytometry (CyTOF) for protein-level detection in rare cell populations

  • Spatial transcriptomics to correlate CDKN2C expression with tissue architecture