CCNA2 Antibody

Basic Research Questions

• What is CCNA2/Cyclin A2 and why is it important in research applications?

  Cyclin A2 (CCNA2) is a member of the highly conserved cyclin family that functions as a critical regulator of the cell cycle. It has a reported amino acid length of 432 and an expected molecular weight of 48.6 kDa, though it is often observed at 54-55 kDa in gel electrophoresis . CCNA2 controls both the G1/S and G2/M transition phases by forming specific serine/threonine protein kinase holoenzyme complexes with cyclin-dependent kinases CDK1 (during G2 and M phase) and CDK2 (during S phase) . Unlike its paralog Cyclin A1 (which is primarily expressed in late pachytene spermatocytes), Cyclin A2 is ubiquitously expressed . It may also be known by alternative names including Cyclin A, CCNA, and CCN1 .

  CCNA2 antibodies are essential tools for studying cell cycle regulation, cancer biology, and cellular proliferation, as aberrant CCNA2 expression is associated with various diseases including retinoblastoma and adenocarcinoma .

• What applications are CCNA2 antibodies commonly used for?

  CCNA2 antibodies are validated for multiple research applications:

  Application	Common Abbreviation	Purpose in CCNA2 Research
  Western Blot	WB	Detection of CCNA2 protein expression levels and molecular weight verification (~49 kDa theoretical, often observed at 54-55 kDa)
  Immunohistochemistry	IHC-P	Visualization of CCNA2 expression in tissue sections, particularly useful for cancer studies
  Immunocytochemistry	ICC	Cellular localization studies of CCNA2 in cultured cells
  Immunofluorescence	IF	High-resolution subcellular localization of CCNA2
  Immunoprecipitation	IP	Isolation of CCNA2 protein complexes to study binding partners
  Flow Cytometry	FCM	Analysis of CCNA2 expression in individual cells, often correlated with cell cycle phase
  ELISA	ELISA	Quantitative measurement of CCNA2 protein levels

  When selecting an antibody, verify that it has been validated for your specific application through published validation data .

• How should species reactivity be considered when selecting a CCNA2 antibody?

  Species reactivity is a critical consideration for experimental design. CCNA2 antibodies vary in their cross-reactivity profiles:

  • Most commercially available antibodies react with human CCNA2

  • Many antibodies cross-react with mouse and rat CCNA2 due to high sequence homology (human Cyclin A2 shares 83% amino acid identity with mouse Cyclin A2 over amino acids 73-199)

  • Some antibodies offer broader reactivity including bovine, monkey (African green monkey), and other species

  Sequence conservation between species may vary across different domains of the protein. When working with non-human models, verify that the epitope recognized by the antibody is conserved in your species of interest. For research involving multiple species, selecting an antibody with validated cross-reactivity can ensure consistency across experiments .

Advanced Research Questions

• How do I determine the optimal CCNA2 antibody dilution for specific applications?

  Determining optimal antibody dilution requires systematic optimization based on application-specific considerations:

  Application	Typical Dilution Range	Optimization Strategy
  Western Blot	1:500-1:2000	Begin with manufacturer's recommendation (e.g., 1:1000) and perform a dilution series. Assess signal-to-noise ratio and adjust accordingly. For weak signals, longer exposure or enhanced chemiluminescence substrates may be preferable to higher antibody concentrations
  IHC-P	1:50-1:200	Start with mid-range dilution and optimize based on staining intensity and background. Antigen retrieval method significantly impacts optimal dilution
  ICC/IF	1:50-1:500	Initial testing at 1:100, then adjust to optimize signal strength while minimizing background. Cell fixation method affects required concentration
  ELISA	~1 μg/ml	Standard curves with recombinant protein help determine optimal concentration for detection range

  Factors influencing optimal dilution include:

  • Antibody affinity and concentration

  • Target protein abundance (CCNA2 expression varies with cell cycle phase)

  • Sample preparation method

  • Detection system sensitivity

  Document optimization results systematically, as optimal conditions may vary between tissue/cell types and experimental conditions .

• What controls should be incorporated when using CCNA2 antibodies?

  Rigorous experimental design requires appropriate controls:

  Positive Controls:

  • Cell lines with known high CCNA2 expression (e.g., HeLa, Jurkat, A549)

  • Tissues with high proliferative capacity

  • Recombinant CCNA2 protein (for Western blot)

  • Cells synchronized in S or G2 phase (CCNA2 expression peaks during these phases)

  Negative Controls:

  • Primary antibody omission control

  • Isotype control (e.g., Mouse IgG2a for monoclonal antibodies like clone CCNA2/2333)

  • Blocking peptide competition (using the immunizing peptide to demonstrate specificity)

  • Tissues/cells with minimal CCNA2 expression (quiescent cells in G0)

  • CCNA2 knockdown/knockout samples if available

  Additional Validation Controls:

  • Multiple antibodies targeting different CCNA2 epitopes

  • Correlation with mRNA expression

  • Cell cycle phase markers to confirm expected expression pattern

  • Pre-adsorption control using recombinant CCNA2 protein

  These controls help ensure that observed signals represent specific CCNA2 detection rather than non-specific binding or artifacts .

• How does CCNA2 expression change during the cell cycle, and how does this affect antibody detection?

  CCNA2 exhibits dynamic expression patterns throughout the cell cycle, creating important considerations for antibody-based detection:

  Cell Cycle Phase	CCNA2 Expression	Subcellular Localization	Detection Considerations
  G0	Low/Absent	N/A	Minimal detection expected; useful as negative control
  Early G1	Low/Absent	N/A	Minimal detection expected
  Late G1/S boundary	Expression begins	Nuclear	Initial detection point
  S phase	Increasing levels	Nuclear	Associates with CDK2; strong nuclear signal
  G2 phase	High levels	Nuclear	Associates with CDK1; strong nuclear signal
  M phase	Decreasing (degraded)	Nuclear/Diffuse	Signal rapidly diminishes during mitotic exit

  Research implications:

  • For studies of CCNA2 function, cell synchronization may be necessary to obtain homogeneous expression

  • Quantitative analysis must account for cell cycle distribution

  • Co-staining with cell cycle markers (e.g., Ki-67, phospho-histone H3) can contextualize CCNA2 expression

  • Fixation timing is critical for capturing transient expression patterns

  • Flow cytometry combined with DNA content analysis provides correlation between CCNA2 levels and cell cycle phase

  CCNA2's cyclic expression pattern means that detection sensitivity varies significantly depending on the cell population's cell cycle distribution .

Methodology and Troubleshooting

• What is the recommended protocol for optimizing Western blot detection of CCNA2?

  Optimizing Western blot for CCNA2 detection requires attention to several critical parameters:

  Sample Preparation:

  • Include protease inhibitors to prevent CCNA2 degradation

  • Phosphatase inhibitors preserve phosphorylation status if studying post-translational modifications

  • Standardize protein loading (30-50 μg total protein per lane is typically sufficient)

  • Denature samples in SDS loading buffer at 95°C for 5 minutes

  Gel Electrophoresis:

  • Use 10% or 5-20% gradient SDS-PAGE gels for optimal resolution around 49-55 kDa

  • Include molecular weight markers spanning 25-75 kDa range

  • Run at 70-90V (as demonstrated in validation experiments)

  Transfer and Detection:

  • Transfer to nitrocellulose membrane at 150 mA for 50-90 minutes

  • Block with 5% non-fat milk or BSA in TBS/PBS

  • Incubate with primary antibody at optimized dilution (typically 1:500-1:2000) overnight at 4°C

  • Wash thoroughly with TBS-0.1% Tween (3-5 times, 5 minutes each)

  • Incubate with appropriate HRP-conjugated secondary antibody (e.g., goat anti-mouse IgG-HRP at 1:10,000)

  • Develop using enhanced chemiluminescence (ECL)

  Troubleshooting Tips:

  • If detecting multiple bands, consider that CCNA2 may present post-translational modifications

  • Expected molecular weight is 49 kDa, but CCNA2 often runs at 54-55 kDa in SDS-PAGE

  • For weak signals, try longer primary antibody incubation, increased antibody concentration, or more sensitive detection reagents

  • High background may require more stringent washing or reduced antibody concentration

  Validated positive controls include HeLa and Jurkat cell lysates, which demonstrate strong CCNA2 expression .

• How can I optimize immunohistochemistry and immunocytochemistry for CCNA2 detection?

  For Immunohistochemistry (IHC-P):

  1. Fixation and Processing:

     • Formalin-fixed paraffin-embedded (FFPE) tissues should be sectioned at 4-6 μm thickness

     • Fresh frozen sections may provide enhanced antigen preservation

  2. Antigen Retrieval:

     • Heat-induced epitope retrieval (HIER) using citrate buffer (pH 6.0) or EDTA buffer (pH 9.0)

     • Enzymatic retrieval using IHC enzyme antigen retrieval reagent for 15 minutes has been successfully validated for some antibodies

  3. Blocking and Antibody Incubation:

     • Block with 10% normal serum from the same species as secondary antibody

     • Incubate with primary antibody at optimal dilution (1:50-1:200) overnight at 4°C

     • For monoclonal antibodies, use biotinylated secondary antibody followed by streptavidin-biotin complex (SABC)

     • Develop with DAB and counterstain with hematoxylin

  For Immunocytochemistry (ICC/IF):

  1. Cell Preparation:

     • Grow cells on coverslips or chamber slides to 70-80% confluence

     • Fix with 4% paraformaldehyde (10-15 minutes) or methanol (-20°C, 10 minutes)

  2. Staining Protocol:

     • Permeabilize with 0.1-0.5% Triton X-100 if using paraformaldehyde fixation

     • Block with 1-5% BSA or 10% normal serum

     • Incubate with primary antibody (1:50-1:500) for 1-2 hours at room temperature or overnight at 4°C

     • For IF, use fluorophore-conjugated secondary antibodies and include DAPI nuclear counterstain

  Optimization Tips:

  • Validate with positive control cell lines (A549, SMMC-7721 cells have been successfully used)

  • When studying cell cycle, consider that CCNA2 expression and localization change throughout the cycle

  • For dual staining, include proliferation markers (Ki67) or cell cycle phase markers to contextualize CCNA2 expression

  • Document exact conditions that yield optimal results for reproducibility

  Both approaches benefit from systematic optimization of fixation, antigen retrieval, and antibody concentration parameters .

• What strategies can help troubleshoot non-specific binding with CCNA2 antibodies?

  Non-specific binding can significantly impact CCNA2 antibody performance. Here are systematic approaches to identify and resolve common issues:

  Common Sources of Non-Specific Binding:

  Issue	Possible Causes	Troubleshooting Strategies
  Multiple bands in Western blot	Post-translational modifications, protein degradation, splice variants, non-specific binding	Use fresh samples with protease inhibitors; optimize blocking conditions; validate with different antibody clones targeting different epitopes; include positive and negative controls
  High background in IHC/ICC	Insufficient blocking, excessive antibody concentration, cross-reactivity, endogenous peroxidase/biotin	Increase blocking time/concentration; titrate antibody; include hydrogen peroxide treatment for endogenous peroxidase quenching; use biotin blocking kit if using biotin-based detection
  Nuclear vs. cytoplasmic staining inconsistency	Fixation artifacts, epitope masking, antibody specificity	Compare different fixation methods; ensure proper permeabilization; validate with subcellular fractionation followed by Western blot; confirm with multiple antibodies

  Experimental Validation Approaches:

  1. Antibody Validation Tests:

     • Peptide competition assays using the immunizing peptide

     • siRNA/shRNA knockdown of CCNA2 to demonstrate reduced signal

     • Testing on known positive and negative tissues/cell lines

     • Comparison with mRNA expression data

  2. Technical Optimization:

     • Test different blocking reagents (BSA, normal serum, commercial blockers)

     • Optimize antibody concentration with dilution series

     • Increase washing stringency (more washes, higher detergent concentration)

     • For immunohistochemistry, test different antigen retrieval methods

  3. Advanced Approaches:

     • Use secondary-only controls to identify background from secondary antibody

     • Include isotype controls matched to primary antibody

     • Pre-adsorb antibody with related proteins to reduce cross-reactivity

     • Consider alternative detection systems

  Documenting all optimization steps systematically creates a valuable reference for troubleshooting recurring issues .

• How can I validate CCNA2 antibody specificity for my experimental system?

  Comprehensive antibody validation ensures reliable experimental results. A multi-tiered approach is recommended:

  Tier 1: Basic Validation

  • Literature review: Examine published validation data for your specific antibody clone

  • Molecular weight verification: Confirm that detected bands match expected size (theoretical 49 kDa, observed 54-55 kDa for CCNA2)

  • Expression pattern analysis: Verify nuclear localization in cycling cells with expected cell-cycle dependent pattern

  Tier 2: Advanced Validation

  • Genetic approaches:

    • siRNA/shRNA knockdown of CCNA2 should reduce antibody signal

    • CRISPR/Cas9 knockout provides definitive negative control

    • Overexpression systems demonstrate signal increase at correct molecular weight

  • Correlation with orthogonal methods:

    • Compare antibody detection with mRNA expression (qPCR, RNA-seq)

    • Use multiple antibodies targeting different CCNA2 epitopes

    • Parallel analysis with mass spectrometry for protein identification

  • Functional validation:

    • Cell cycle synchronization to demonstrate expected expression patterns

    • Treatment with CDK inhibitors to observe impact on CCNA2-CDK complexes

    • Co-immunoprecipitation to verify known interaction partners (CDK1, CDK2)

  Tier 3: Application-Specific Validation

  • For IHC: Compare staining patterns across tissue types with known CCNA2 expression

  • For WB: Include positive controls (HeLa, Jurkat cells) and negative controls

  • For ICC/IF: Correlation with other cell cycle markers and DNA content

  • For IP: Verify that immunoprecipitated protein is recognized by a different CCNA2 antibody

  Documentation Recommendations:

  • Record complete antibody information: manufacturer, catalog number, lot number, clone ID, host species

  • Document all validation experiments with appropriate controls

  • Include antibody validation data in publications and presentations

  This comprehensive validation approach ensures experimental reproducibility and supports meaningful interpretation of CCNA2-related findings .