CCNDBP1 Antibody

What is CCNDBP1 and why is it significant in academic research?

CCNDBP1 (Cyclin D1 binding protein 1), also known as DIP1, GCIP, or HHM, is a helix-loop-helix protein that lacks a DNA-binding region . It has gained significance in research due to its diverse biological functions:

• It's expressed in various tissues including thymus, spleen, liver, small intestine, colon, brain, muscle, heart, kidney, lung, and peripheral leukocytes

• It functions as a tumor suppressor in multiple cancers including liver, breast, gastric, lung, prostate, and colon cancers

• It interacts with various proteins including cyclin D1, SYF2, E12, CT847, Jab1, Sirt6, MyoD, and Olig1

• It plays roles in G1/S cell cycle phase progression in hepatocytes

• It's involved in the DNA damage response pathway and chemoresistance mechanisms

• It functions as a positive regulator of skeletal myogenesis

This multifunctional nature makes CCNDBP1 a valuable target for studying cancer mechanisms, DNA damage response, and muscle development.

The expected molecular weight for CCNDBP1 is approximately 40-40.3 kDa , though variations may occur:

• The calculated molecular weight based on amino acid sequence is 40.3 kDa

• The protein contains 360 amino acids

• Some researchers have observed bands at different molecular weights:

  • A band at approximately 55 kDa has been reported in K562 lysate using certain antibodies

  • The discrepancy between predicted (41 kDa and 27 kDa) and observed bands requires careful validation

When performing western blots, include positive controls from validated cell lines such as HeLa cells, which have been confirmed to express CCNDBP1 . Always validate new antibodies with known controls to ensure proper detection of the target protein.

What are the optimal protocols for CCNDBP1 antibody applications in western blot?

For optimal western blot results with CCNDBP1 antibodies:

Sample Preparation:

• Extract total protein from cells or tissues using standard lysis buffers

• For cells, protocols similar to those used for HLE and HepG2 cell lines are recommended

• Include protease inhibitors to prevent degradation

Western Blot Protocol:

• Load 20-50 μg of total protein per lane

• Use 10-12% SDS-PAGE gels for optimal separation

• Transfer proteins to PVDF or nitrocellulose membranes

• Block with 5% non-fat milk or BSA in TBST

Antibody Dilutions:

• Primary antibody: 1:500-1:1000 dilution is recommended for most CCNDBP1 antibodies

• Use antibodies validated for western blot applications, such as those from Proteintech (12363-1-AP)

• For anti-CCNDBP1 antibody from Abcam (ab220275), a 1:2000 dilution has been reported effective

Detection:

• Follow with appropriate HRP-conjugated secondary antibody

• Visualize using chemiluminescence detection systems

• Use β-actin or GAPDH as loading controls

What are the recommended protocols for immunohistochemistry with CCNDBP1 antibodies?

For effective immunohistochemistry staining of CCNDBP1:

Tissue Preparation:

• Use formalin-fixed, paraffin-embedded tissue sections (4-6 μm thickness)

• For optimal results with CCNDBP1 antibodies, antigen retrieval is critical

Antigen Retrieval Methods:

• Primary recommendation: TE buffer pH 9.0

• Alternative method: Citrate buffer pH 6.0

• Heat-induced epitope retrieval (HIER) is generally preferred

Staining Protocol:

• Block endogenous peroxidase activity with 3% H₂O₂

• Block non-specific binding with serum-based blocking buffer

• Apply primary antibody at 1:20-1:200 dilution or 3-6 μg/ml for IHC

• Incubate at 4°C overnight for optimal results

• Follow with appropriate detection system (e.g., HRP-DAB)

Visualization and Analysis:

• Counterstain with hematoxylin

• CCNDBP1 shows textured cytoplasmic staining in human tissues

• In prostate tissues, emphasis on the luminal side of secretory cells in glands has been observed

• Analysis can be performed using AOD (average optical density) value measurements

How can I validate the specificity of a CCNDBP1 antibody?

Validating antibody specificity is crucial for reliable results. For CCNDBP1 antibodies:

Positive Controls:

• Use cell lines with confirmed CCNDBP1 expression, such as:

  • HeLa cells (validated for many CCNDBP1 antibodies)

  • HLE and HepG2 cell lines (used in published studies)

Negative Controls:

• Use CCNDBP1 knockout models when available

• Employ siRNA or shRNA knockdown of CCNDBP1 in cell lines

  • The C1 and C2 shRNAs have been successfully used with >70% knockdown efficiency

Specificity Tests:

• Peptide competition assay: Pre-incubate the antibody with immunizing peptide before application

  • A successfully blocked 55 kDa band has been reported in K562 lysate

• Compare multiple antibodies targeting different epitopes

• Use overexpression systems with tagged CCNDBP1 constructs

Testing Across Applications:

• Cross-validate results using different detection methods (WB, IHC, IF)

• Confirm subcellular localization patterns match known distribution patterns

How can CCNDBP1 antibodies be used to study the DNA damage response pathway?

CCNDBP1 plays a critical role in the DNA damage response through the ATM-CHK2 pathway. To investigate this:

Experimental Approach:

• Radiation/Chemotherapy Models:

  • Treat cells with X-ray radiation (0.8 Gy/min, 3 min) or cisplatin (20 μM CDDP)

  • Detect CCNDBP1 expression changes via western blot

  • Compare CCNDBP1-overexpressing cells with controls for survival rates

• ATM-CHK2 Pathway Analysis:

  • Use antibodies against key proteins in this pathway alongside CCNDBP1:

    • ATM (ab78, 1:2000 dilution)

    • phospho-S1981 ATM (ab36810, 1:1000)

    • CHK2 (1:1000)

    • phospho-CHK2 (Thr68) (1:1000)

• EZH2 Inhibition Studies:

  • Investigate the relationship between CCNDBP1 and EZH2 (ab186006, 1:1000)

  • Design experiments to confirm that CCNDBP1 activates the ATM-CHK2 pathway through EZH2 inhibition

• Functional Assays:

  • Cell growth assays using MTT or WST-1 after DNA damage induction

  • Track DNA repair efficiency in CCNDBP1-expressing versus control cells

Data Analysis Framework:

• Compare growth rates and survival after DNA damage in cells with different CCNDBP1 expression levels

• Quantify phosphorylation levels of ATM and CHK2 in relation to CCNDBP1 expression

• Analyze the correlation between CCNDBP1, EZH2 levels, and DNA damage resistance

What is the role of CCNDBP1 in skeletal myogenesis and how can antibodies help study this process?

CCNDBP1 functions as a positive regulator of skeletal myogenesis. To investigate this role:

Experimental Design:

• Differentiation Studies:

  • Monitor CCNDBP1 expression during C2C12 myoblast differentiation

  • Compare differentiation efficiency in CCNDBP1-overexpressing, knockdown, and control cells

  • Assess morphological changes and myotube formation

• Marker Analysis:

  • Use antibodies against myogenic markers alongside CCNDBP1:

    • Myosin Heavy Chain (MHC) - terminal differentiation marker

    • Myogenin - early differentiation marker

    • MyoD - myogenic determination factor

• Protein Interaction Studies:

  • Investigate CCNDBP1 interaction with MyoD using co-immunoprecipitation

  • Analyze how this interaction enhances MyoD binding to target genes

  • Study the N-terminus and HLH domain requirements for this interaction

• Functional Assays:

  • Differentiation index: Percentage of MHC-positive cells

  • Fusion index: Percentage of multinucleated myotubes

  • qRT-PCR for myogenic markers (Myh1, myogenin)

In Vivo Approaches:

• Study muscle development in CCNDBP1 knockout mice

• Measure cross-sectional area of skeletal muscles (e.g., tibialis anterior)

• Assess muscle regeneration ability and grip strength

How can CCNDBP1 antibodies be used to investigate its tumor suppressor function?

CCNDBP1 has been identified as a tumor suppressor in multiple cancers. To study this function:

Experimental Approaches:

Data from Dedifferentiated Liposarcoma (DDL) Study:

• Low CCNDBP1 expression associated with poor prognosis

• CCNDBP1 identified as an independent prognostic factor for PFS

• CCNDBP1 expression regulated by DNA methylation

• CCNDBP1 inhibits EMT, reducing malignant behaviors of cancer cells

Why might I see inconsistent band sizes when using CCNDBP1 antibodies in western blot?

Inconsistent band sizes are a common challenge with CCNDBP1 detection:

Potential Causes and Solutions:

Documented Variations:

• Expected molecular weight: ~40.3 kDa based on amino acid sequence

• Observed band at ~55 kDa in K562 lysate

• Scientific literature currently lacks clear explanation for the discrepancy between predicted (41 kDa and 27 kDa) and observed bands

When troubleshooting, always include positive controls from validated cell lines (e.g., HeLa cells) and consider using CCNDBP1 knockout/knockdown samples as negative controls .

How should I interpret changes in CCNDBP1 expression in experimental contexts?

Interpreting CCNDBP1 expression changes requires understanding its complex roles in different cellular contexts:

Context-Dependent Interpretation:

Quantification Methods:

• Western blot: Normalize CCNDBP1 band intensity to loading controls (β-actin, GAPDH)

• IHC: Use average optical density (AOD) measurements for quantitative comparison

• qRT-PCR: Utilize the 2^-ΔΔCt method with appropriate housekeeping genes

What contradictions exist in the literature regarding CCNDBP1 function, and how should researchers approach them?

The CCNDBP1 literature contains several apparent contradictions that researchers should be aware of:

Key Contradictions and Reconciliation Approaches:

• Tumor Suppressor vs. Pro-Survival Function:

  • Traditional view: CCNDBP1 is a tumor suppressor in multiple cancers

  • Contradicting evidence: CCNDBP1 can promote cancer cell survival by rescuing from DNA damage

  • Reconciliation approach: Study context-specific functions; CCNDBP1 may have dual roles depending on cellular environment and cancer type

• Growth Regulation:

  • Contradiction: CCNDBP1 overexpression increased growth ratio in HCC cells but inhibited proliferation in DDL cells

  • Approach: Investigate tissue-specific effects and pathway interactions; differentiate between effects on normal growth vs. recovery from damage

• Mechanistic Pathway Interactions:

  • Different studies highlight interactions with different pathways (Cyclin D1/CDK4/RB1/E2F pathway vs. ATM-CHK2 pathway )

  • Integration approach: Consider CCNDBP1 as a multifunctional protein involved in multiple cellular pathways

Research Design Recommendations:

• Include multiple cell lines/tissues to capture context-dependent effects

• Perform both gain- and loss-of-function experiments (overexpression and knockdown)

• Correlate in vitro findings with in vivo models and clinical data

• Use multiple technical approaches (WB, IHC, functional assays) to build comprehensive understanding

How can CCNDBP1 antibodies be utilized in studying DNA methylation regulation of gene expression?

DNA methylation appears to regulate CCNDBP1 expression, offering opportunities for epigenetic research:

Methodological Approach:

• Methylation Site Analysis:

  • Focus on key methylation sites identified for CCNDBP1, such as cg05194114 and cg22184989

  • Use bisulfite sequencing or methylation-specific PCR to analyze methylation status

  • Correlate methylation levels with CCNDBP1 protein expression via antibody detection

• Demethylation Studies:

  • Treat cells with demethylating agents (e.g., 5-azacytidine)

  • Monitor changes in CCNDBP1 expression using antibodies in western blot or IHC

  • Compare expression patterns before and after treatment

• Clinical Correlation:

  • Analyze patient samples for methylation status and protein expression

  • Investigate the relationship between methylation, CCNDBP1 levels, and clinical outcomes

  • Consider developing predictive biomarkers based on these relationships

Research Applications:

• Development of epigenetic therapies targeting CCNDBP1 methylation

• Identification of patients who might benefit from demethylating agents

• Understanding the upstream regulation of CCNDBP1 in different disease contexts

What are the future directions for CCNDBP1 research in inflammatory diseases?

Emerging evidence suggests CCNDBP1 may play a role in inflammatory conditions, particularly colitis:

Research Framework:

• DSS-Induced Colitis Model:

  • Study the contribution of Ccndbp1 and the Atm-Chk2 pathway in colitis models

  • Compare wild-type and Ccndbp1-knockout mice responses to DSS treatment

  • Analyze inflammatory markers (IFN-γ, IL-1β, IL-6, IL-10) in relation to Ccndbp1 expression

• Pathway Analysis:

  • Investigate the crosstalk between DNA damage response pathways and inflammatory signaling

  • Study how Ccndbp1 regulates inflammatory gene expression

  • Explore potential therapeutic interventions targeting this pathway

• Translational Research:

  • Examine CCNDBP1 expression in human inflammatory bowel disease samples

  • Correlate expression levels with disease severity and treatment response

  • Evaluate CCNDBP1 as a potential biomarker for inflammatory conditions

Experimental Design Considerations:

• Use tissue-specific conditional knockout models to isolate effects

• Employ both genetic and pharmacological approaches to modulate the pathway

• Consider sex-specific differences in inflammatory responses and CCNDBP1 function

The study of CCNDBP1 in inflammatory contexts represents an emerging field that may connect DNA damage response pathways with inflammatory disease mechanisms, potentially offering new therapeutic targets.