CCND2 Antibody

What is the biological function of CCND2 protein?

CCND2 serves as a regulatory component of the cyclin D2-CDK4 complex that phosphorylates and inhibits retinoblastoma (RB) protein family members, controlling cell-cycle progression during G1/S transition. It hypophosphorylates RB1 in early G1 phase and functions as a major integrator of various mitogenic and antimitogenic signals . CCND2 belongs to the highly conserved cyclin family characterized by dramatic periodicity in protein abundance throughout the cell cycle .

How should I select the appropriate CCND2 antibody for my experiment?

Selection should be based on:

• Application compatibility: Verify antibody validation for your specific application (WB, IHC, IF/ICC, Flow)

• Species reactivity: Ensure reactivity with your experimental model (human, mouse, rat)

• Epitope recognition: Consider antibodies targeting different protein regions for confirmation

• Clonality: Monoclonal for specificity; polyclonal for broader epitope detection

• Validation data: Review published applications and knockout/knockdown validation

Always validate antibodies in your specific experimental system, as performance may vary across tissues and cell types .

What is the molecular weight of CCND2 and how does this inform antibody validation?

CCND2 has a calculated molecular weight of approximately 32-33.1 kDa, with observed molecular weight typically around 34 kDa in Western blots . When validating antibodies, note that:

• Post-translational modifications may cause slight shifts in observed weight

• Multiple bands may indicate isoforms, degradation products, or non-specific binding

• Validation should include positive control tissues/cells known to express CCND2 (e.g., Caco-2, MCF-7, NIH/3T3, U2OS, HEK-293 cells)

• Knockout/knockdown validation provides definitive evidence of specificity

For instance, ab207604 was validated using CCND2 knockout cells, which showed loss of signal at the expected molecular weight, though additional cross-reactive bands were observed in both wild-type and knockout samples .

What are optimal protocols for CCND2 immunohistochemistry (IHC)?

For effective CCND2 IHC:

• Fixation: 10% neutral buffered formalin for 24-48 hours

• Antigen retrieval:

  • Primary option: TE buffer pH 9.0 (recommended by Proteintech)

  • Alternative: Citrate buffer pH 6.0

• Blocking:

  • Standard: 3% BSA, 10% Donkey Serum in TBS with 0.1% Tween-20

  • For enhanced specificity: Add 5% nonfat milk to blocking buffer

• Primary antibody incubation: Dilute 1:250-1:1000 (application-dependent), incubate overnight at 4°C

• Detection system: For higher sensitivity, use biotin-streptavidin amplification systems

  • Example protocol: Incubate in biotinylated secondary antibody (1:400, 1 hour), then peroxidase-conjugated tertiary antibody (1 hour), followed by DAB reaction

• Controls: Include positive control tissues (renal cell carcinoma, gliomas) and negative controls (omit primary antibody)

How can I optimize Western blot protocols for CCND2 detection?

For optimal CCND2 Western blot results:

• Sample preparation:

  • Heat samples to 70°C for 10 minutes with LDS Sample Buffer

  • Use NuPAGE® 4-12% Bis-Tris gels for optimal separation

• Membrane blocking:

  • Standard: 5% dried skimmed milk in PBS with 0.1% Tween-20 (1 hour at room temperature)

  • Enhanced specificity: 5% non-fat dry milk in TBS-T (0.05% Tween 20)

• Antibody dilutions and incubation:

  • Primary antibody: 1:200-1:2000 dilution (overnight at 4°C)

  • Secondary antibody: 1:10,000 dilution (1 hour at room temperature)

• Detection systems:

  • Chemiluminescent: SuperSignal West Femto Substrate provides high sensitivity

  • Fluorescent: LI-COR system enables quantitative analysis with secondary antibodies such as IRDye 800CW and IRDye 680LT

• Quantification:

  • Normalize CCND2 expression to loading controls (β-actin, GSK-3β)

  • For accurate quantification, establish linear detection range through serial dilutions

What controls should I include when using CCND2 antibodies in my experiments?

Include these critical controls:

• Positive controls:

  • Cell lines with confirmed CCND2 expression (Caco-2, MCF-7, NIH/3T3, U2OS, HEK-293)

  • Tissue samples known to express CCND2 (renal cell carcinoma, gliomas)

• Negative controls:

  • CCND2 knockout/knockdown cells (validated in several studies)

  • Primary antibody omission

  • Isotype controls

• Loading/technical controls:

  • Housekeeping proteins (β-actin, vinculin, GSK-3β)

  • For protein degradation assessment: include both N- and C-terminal targeting antibodies

• Signal specificity controls:

  • Phospho-specific antibody controls: Use phosphatase treatment

  • Peptide competition assays to confirm epitope specificity

  • Multiple antibodies targeting different epitopes

How can I use CCND2 antibodies to study developmental disorders and megalencephaly?

CCND2 mutations have been linked to megalencephaly-polymicrogyria-polydactyly-hydrocephalus (MPPH) syndrome. For such studies:

• Mutation-specific approaches:

  • Use antibodies recognizing specific mutations (p.Thr280Ala, p.Pro281Arg, p.Val284Gly)

  • Compare wild-type vs. phosphodeficient (p.Thr280Ala) vs. phosphomimetic (p.Thr280Asp) CCND2 forms

• Cell cycle analysis:

  • Co-stain with phosphohistone H3 (PH3) to identify cells in M-phase

  • Quantify mitotic index in cells expressing wild-type vs. mutant CCND2

• Signaling pathway investigation:

  • Analyze CCND2 interaction with CDK4 through co-immunoprecipitation

  • Examine phosphorylation status of downstream targets like retinoblastoma protein

  • Investigate AKT/mTOR pathway activation using phospho-specific antibodies (pAKT, pS6)

• In vivo developmental studies:

  • Use electroporation of GFP-tagged CCND2 constructs followed by immunohistochemical analysis

  • Examine proliferation in ventricular/subventricular zones using CCND2 antibodies

What approaches can I use to investigate CCND2's role in cardiac regeneration?

Recent research has explored CCND2's potential in cardiac regeneration:

• Cardiomyocyte-specific expression systems:

  • Use the CCND2-cardiomyocyte-specific modified mRNA translation system (cardiomyocyte SMRTs)

  • This system utilizes microRNA recognition elements (miR-1, miR-208) for cardiomyocyte-specific expression

• Proliferation markers:

  • Co-stain with PH3 to identify mitotic cardiomyocytes

  • Use Aurora B (AuB) with distinct staining patterns to differentiate between:

    • Asymmetrical AuB expression (karyokinesis within a single cell)

    • Symmetrical AuB expression (cytokinesis/cell division)

• Hypertrophy vs. proliferation assessment:

  • Combine CCND2 staining with cardiomyocyte size measurements

  • Distinguish between multinucleation events and true cell division

• Therapeutic delivery systems:

  • Evaluate modified mRNA translation systems for targeted CCND2 delivery

  • Compare with controls expressing other proteins (luciferase, GFP) or delivery vehicle alone

How can CCND2 antibodies be used in cancer research?

CCND2 has complex roles in cancer development and progression:

What causes multiple bands in CCND2 Western blots and how should I interpret them?

Multiple bands may appear due to:

• Post-translational modifications:

  • Phosphorylation at sites like Thr280 affects protein stability and function

  • Different phosphorylation states may appear as distinct bands

• Protein degradation:

  • CCND2 undergoes regulated degradation during cell cycle

  • Lower molecular weight bands may represent degradation products

• Cross-reactivity:

  • Some CCND2 antibodies show cross-reactivity with related proteins

  • Example: ab207604 showed additional cross-reactive bands in both wild-type and CCND2 knockout cells

• Splice variants:

  • Alternative splicing may generate different CCND2 isoforms

Interpretation approach:

• Confirm specificity using knockout/knockdown controls

• Compare band patterns across multiple antibodies targeting different epitopes

• Use phospho-specific antibodies to identify specific modifications

• Consider performing mass spectrometry to identify ambiguous bands

How can I resolve weak or absent CCND2 signal in my experiments?

For weak or absent signals:

• Antibody selection and handling:

  • Verify antibody viability (check for precipitation, proper storage)

  • Consider trying antibodies from different suppliers/clones

  • Some antibodies perform better in specific applications (WB vs. IHC vs. IF)

• Sample preparation optimization:

  • For proteins with low abundance: increase loading amount or use enrichment techniques

  • Optimize lysis buffers to ensure complete protein extraction

  • For tissue samples: ensure proper fixation (overfixation can mask epitopes)

• Protocol modifications:

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

  • Increase antibody concentration (within recommended range)

  • For IHC/IF: Test different antigen retrieval methods (pH 9.0 TE buffer vs. pH 6.0 citrate buffer)

  • For WB: Try different blocking reagents (BSA vs. milk)

• Detection system enhancement:

  • Use more sensitive detection reagents (SuperSignal West Femto)

  • For IHC: Implement signal amplification (biotin-streptavidin system)

  • For IF: Use brighter fluorophores or tyramide signal amplification

How should I interpret varying levels of CCND2 expression across different tissues and cell types?

CCND2 expression varies naturally across tissues and cellular contexts:

• Cell cycle-dependent expression:

  • CCND2 shows "dramatic periodicity in protein abundance through the cell cycle"

  • Synchronize cells to compare equivalent cell cycle phases

• Tissue-specific regulation:

  • CCND2 is differentially expressed across tissues (blood, brain, bone, ovary, muscle, heart, lung, liver)

  • Compare to published expression data for your tissue of interest

• Disease state alterations:

  • CCND2 expression changes in pathological conditions

  • Example: Downregulated in lung adenocarcinoma compared to normal tissue

  • Associated with various neoplasms (breast, prostate, lung, ovarian)

• Quantification approach:

  • Use appropriate normalization strategies for accurate comparison

  • Establish relative quantification through serial dilutions within linear range

  • Compare both RNA (qPCR) and protein levels to distinguish transcriptional vs. post-transcriptional regulation

How can phospho-specific CCND2 antibodies advance our understanding of protein regulation?

Phosphorylation plays a crucial role in CCND2 stability and function:

• Key phosphorylation sites:

  • Thr280 phosphorylation affects protein stability and function

  • Phosphodeficient mutants (p.Thr280Ala) show increased stabilization

• Research applications:

  • Compare phosphorylated vs. non-phosphorylated CCND2 levels across cell cycle

  • Investigate upstream kinases through inhibitor studies

  • Examine phosphorylation status in disease states

• Available tools:

  • Phospho-specific antibodies (e.g., Anti-Phospho-Cyclin D2 (T280) from BosterBio)

  • Phosphomimetic (p.Thr280Asp) and phosphodeficient (p.Thr280Ala) constructs for functional studies

• Methodological considerations:

  • Include phosphatase inhibitors during protein extraction

  • Use phosphatase treatment as negative control

  • Consider 2D gel electrophoresis to separate phospho-isoforms

What novel approaches are being developed for studying CCND2 in tissue-specific contexts?

Emerging techniques for tissue-specific CCND2 studies:

• Targeted mRNA delivery systems:

  • Specific modified mRNA translation systems (SMRTs) allow tissue-specific expression

  • Example: Cardiomyocyte-specific CCND2 expression using microRNA recognition elements

• Single-cell analysis:

  • Single-cell RNA-seq combined with antibody-based protein detection

  • Spatial transcriptomics with immunohistochemistry for location-specific expression patterns

• In vivo imaging:

  • Live-cell imaging using fluorescently tagged CCND2 constructs

  • Correlate with immunostaining using CCND2 antibodies for validation

• Organ-on-chip technologies:

  • Study CCND2 dynamics in controlled microenvironments mimicking tissue architecture

  • Apply tissue-specific mechanical forces while monitoring CCND2 expression/localization

How might CCND2 research contribute to therapeutic developments?

CCND2 research has significant therapeutic implications:

• Cardiac regeneration:

  • CCND2-cardiomyocyte SMRTs can induce cardiomyocyte proliferation after myocardial infarction

  • This approach has shown promise in both mouse and pig models of cardiac injury

• Cancer therapeutics:

  • CCND2 serves as a prognostic biomarker in lung adenocarcinoma

  • Understanding CCND2's role in immune infiltration could inform immunotherapy strategies

• Developmental disorders:

  • CCND2 mutations are associated with megalencephaly-polymicrogyria-polydactyly-hydrocephalus syndrome

  • Understanding mutation effects could lead to targeted treatments

• Cell cycle regulation:

  • CCND2 interactions with CDK4, CDK6, and other cell cycle regulators represent potential therapeutic targets

  • Inhibitors targeting these interactions are being explored for various conditions