CDCA7 Antibody

What is CDCA7 and why is it significant for research?

CDCA7 (cell division cycle associated 7) is a nuclear protein that plays multiple important roles in cellular processes including MYC-mediated cell transformation, apoptosis, and transcriptional regulation. It has a calculated molecular weight of approximately 43 kDa and is observed at 40-43 kDa in experimental analysis . CDCA7 has gained significant research attention due to its involvement in multiple biological processes:

• Critical role in DNA methylation at juxtacentromeric regions through its interaction with the chromatin remodeler HELLS

• Mutations in CDCA7 are associated with Immunodeficiency-Centromeric instability-Facial anomalies (ICF) syndrome

• Enhanced expression in various cancers, particularly pancreatic cancer and Burkitt's lymphoma

• Involvement in hematopoietic stem cell (HSC) emergence as a Notch transcriptional target

Research on CDCA7 spans multiple disciplines including cancer biology, epigenetics, immunology, and developmental biology, making it a versatile target for investigation.

What types of CDCA7 antibodies are currently available for research?

Several types of CDCA7 antibodies are available for research applications:

What are the optimal protocols for using CDCA7 antibodies in Western blotting?

For Western blotting with CDCA7 antibodies, the following methodological approach is recommended:

• Sample preparation:

  • CDCA7 has been successfully detected in various cell lines including A549, HeLa, HEK-293, and Raji cells

  • Use standard protein extraction protocols with protease inhibitors

• Recommended dilution ranges:

  • For polyclonal antibodies such as Proteintech 15249-1-AP: 1:1000-1:5000

  • For custom antibodies: validation-dependent, typically 1:500-1:1000

• Detection optimization:

  • CDCA7 protein typically appears at 40-43 kDa

  • Both LI-COR Odyssey infrared detection systems and HRP-conjugated secondary antibodies with ECL detection have been successfully used

  • For infrared detection: use IRDye secondary antibodies at manufacturer-recommended dilutions in Odyssey blocking buffer

  • For chemiluminescence: HRP-conjugated secondary antibodies with standard ECL reagents

• Isoform considerations:

  • Be aware that multiple CDCA7 isoforms exist (up to 6 reported), with the most commonly detected being CDCA7-2 in cancer cells

  • The canonical isoform is 371 amino acids with a mass of 42.6 kDa

For optimal results, validation experiments should include positive controls using cell lines known to express CDCA7 (e.g., BL cell lines for CDCA7-2) and negative controls such as CDCA7 knockout cell lines or antibody neutralization with immunizing peptide .

How can I optimize CDCA7 antibody use for immunofluorescence studies?

For successful immunofluorescence (IF) applications with CDCA7 antibodies, consider the following protocol optimizations:

• Cell selection and preparation:

  • HepG2 cells have been validated for CDCA7 IF applications

  • Use standard 4% paraformaldehyde fixation (10-15 minutes at room temperature)

  • Permeabilize with 0.1-0.5% Triton X-100 in PBS (5-10 minutes)

• Antibody dilution and incubation:

  • For primary antibody: dilution range of 1:10-1:100 for commercial polyclonal antibodies

  • Use overnight incubation at 4°C for optimal signal-to-noise ratio

  • For secondary antibodies: typically 1:200-1:500 dilution, 1 hour at room temperature

• Visualization strategies:

  • CDCA7 shows two distinct localization patterns:

    • Diffuse nuclear distribution in most cell types

    • Concentrated in constitutive heterochromatin foci during S phase

  • Co-staining with cell cycle markers or EdU pulse labeling can help identify S-phase cells with characteristic CDCA7 foci

  • Co-staining with H3K9me3 antibodies helps identify constitutive heterochromatin regions

• Controls and validation:

  • Include wild-type and CDCA7-mutant cells (especially ICF syndrome mutations like R285C in mouse or R304C in human) to demonstrate specificity of heterochromatin localization patterns

  • DAPI staining helps visualize the DAPI-bright spots that correlate with CDCA7 heterochromatin localization during S phase

Research has shown that CDCA7's localization changes during the cell cycle, with enrichment in constitutive heterochromatin during DNA replication, which is disrupted by ICF syndrome mutations in the cysteine-rich domain (CRD) . This dynamic localization should be considered when designing and interpreting IF experiments.

How do CDCA7 antibodies help investigate the protein's role in DNA methylation maintenance?

CDCA7 antibodies have been instrumental in elucidating the protein's critical role in DNA methylation maintenance, particularly at heterochromatic regions:

• Chromatin immunoprecipitation (ChIP) applications:

  • CDCA7 antibodies can be used to identify genomic regions bound by CDCA7, particularly at juxtacentromeric satellite regions

  • The protein preferentially binds to hemimethylated DNA with strand-specific CpG methylation, particularly in non-B DNA structures formed during replication

  • ChIP-seq analysis has shown enrichment at centromeres of human chromosomes

• Co-localization studies:

  • Immunofluorescence with CDCA7 antibodies reveals co-localization with HELLS at heterochromatin domains during S-phase

  • CDCA7 is required for the recruitment of HELLS to centromeric heterochromatin, and this can be visualized using dual antibody staining

• Protein complex identification:

  • Immunoprecipitation with CDCA7 antibodies followed by mass spectrometry has identified interaction partners including HELLS

  • Co-immunoprecipitation experiments demonstrate that CDCA7 interacts with HELLS via N-terminal alpha helices

• Functional complementation analysis:

  • In CDCA7 knockout systems, antibodies can validate the expression of reintroduced wild-type or mutant CDCA7 proteins (e.g., ICF syndrome mutations) to analyze their impact on DNA methylation patterns

  • Combining antibody detection with DNA methylation analysis techniques (bisulfite sequencing, methylation-sensitive restriction enzyme assays) helps correlate CDCA7 function with methylation status

Recent research has demonstrated that CDCA7 functions as a hemimethylated DNA sensor through its C-terminal 4CXXC-type zinc finger domain, which recognizes CpG dyads in non-B DNA structures . This sensing activity is critical for recruiting the HELLS chromatin remodeler to facilitate DNA methylation maintenance at heterochromatic regions, particularly during DNA replication.

What role does CDCA7 play in cancer progression and how can antibodies help investigate this?

CDCA7 has emerged as a significant factor in cancer biology, particularly in promoting cancer cell proliferation, invasion, and therapy resistance. CDCA7 antibodies serve as essential tools for investigating these roles:

• Expression level analysis in tumors:

  • Western blotting and immunohistochemistry (IHC) with CDCA7 antibodies have demonstrated upregulation in multiple cancer types

  • In Burkitt's lymphoma cell lines and tumor biopsies, CDCA7-2 isoform shows markedly higher expression compared to lymphoblastoid cell lines

  • In pancreatic cancer, CDCA7 overexpression correlates with enhanced proliferation, migration, invasion, and gemcitabine resistance

• Mechanistic pathway investigation:

  • Antibodies help establish CDCA7's role in STAT3 transcriptional regulation in pancreatic cancer

  • Immunoprecipitation experiments with CDCA7 antibodies can identify interacting partners in cancer-specific signaling pathways

  • CDCA7's impact on aerobic glycolysis can be assessed by correlating CDCA7 levels (via antibody detection) with hexokinase 2 expression and glycolytic activity

• Therapeutic response monitoring:

  • CDCA7 antibodies can be used to monitor expression changes in response to cancer therapies

  • In gemcitabine-resistant pancreatic cancer, CDCA7 levels correlate with resistance mechanisms via enhanced glycolysis

• Histopathological applications:

  • IHC with CDCA7 antibodies (recommended dilution 1:250-1:1000) has been validated for human colon cancer tissue using TE buffer pH 9.0 for antigen retrieval

  • This approach could be extended to tissue microarrays for high-throughput analysis of CDCA7 expression across tumor types and grades

The emerging role of CDCA7 in metabolic reprogramming of cancer cells, particularly through enhanced aerobic glycolysis, suggests it may serve as both a biomarker and therapeutic target . Antibody-based detection methods are fundamental to exploring these translational applications in cancer research.

How can I validate the specificity of CDCA7 antibodies for my research?

Validating CDCA7 antibody specificity is crucial for reliable research outcomes. Several complementary approaches are recommended:

• Genetic validation approaches:

  • Use CDCA7 knockout cell lines generated by CRISPR/Cas9 gene editing as negative controls

  • Compare signal in wild-type versus knockout samples across applications (Western blot, IF, IHC)

  • Alternatively, use siRNA or shRNA knockdown to demonstrate signal reduction corresponding to reduced CDCA7 expression

• Peptide competition assays:

  • Pre-incubate the CDCA7 antibody with excess immunizing peptide before application

  • The specific signal should be neutralized or significantly reduced, as demonstrated with the S99 antibody in BL cell lysates

  • Include non-competing peptide controls to confirm specificity of the competition

• Recombinant protein controls:

  • Express tagged recombinant CDCA7 (both isoforms if relevant) in expression systems

  • Demonstrate that the antibody recognizes the correct molecular weight bands

  • For example, validation using CDCA7-1 and CDCA7-2 ectopically expressed in HEK-293T cells has confirmed antibody specificity

• Cross-validation with multiple antibodies:

  • Compare results using different antibodies targeting distinct epitopes of CDCA7

  • Concordant results across antibodies increase confidence in specificity

  • Note that some studies have reported reproducibility issues with commercial antibodies, highlighting the importance of thorough validation

• Application-specific controls:

  • For IF: demonstrate expected nuclear localization and cell-cycle dependent patterns

  • For IHC: include tissue samples known to express varying levels of CDCA7 (e.g., thymus and small intestine show higher expression)

  • For IP: confirm enrichment of known CDCA7 interaction partners like HELLS

Given the reported variability in commercial antibody performance , researchers may consider developing their own validated antibodies for critical applications, particularly when studying specific CDCA7 isoforms or when working with challenging samples.

What are common pitfalls when using CDCA7 antibodies and how can they be overcome?

Researchers working with CDCA7 antibodies may encounter several challenges that can affect experimental outcomes. Here are common pitfalls and strategies to overcome them:

• Isoform detection challenges:

  • Pitfall: Failure to detect specific CDCA7 isoforms (up to 6 reported variants)

  • Solution: Verify which isoforms your antibody targets; the S99 antibody detects both CDCA7-1 and CDCA7-2 but shows higher sensitivity for CDCA7-2 in BL cells

  • Approach: Use positive controls expressing specific isoforms and select antibodies raised against common regions if multiple isoform detection is desired

• Cell-cycle dependent localization:

  • Pitfall: Inconsistent immunofluorescence patterns due to CDCA7's dynamic localization

  • Solution: Synchronize cells or co-stain with cell cycle markers; CDCA7 shows distinct localization patterns (constitutive heterochromatin foci) specifically during S phase

  • Approach: For heterochromatin localization studies, analyze cells 12-16 hours after release from serum starvation when approximately 43% of cells are in S phase

• Buffer compatibility issues:

  • Pitfall: Suboptimal signal in different applications due to buffer incompatibility

  • Solution: Use optimized buffers; for Western blotting, different antibodies perform best in specific buffers:

    • LI-COR Odyssey blocking buffer for some applications

    • 5% milk in PBS-T for others

    • Signal enhancement solutions (e.g., Toboyo Can Get Signal) for challenging detections

• Background signal in immunofluorescence:

  • Pitfall: High background obscuring specific CDCA7 localization patterns

  • Solution: Optimize blocking (3-5% BSA or normal serum), increase washing steps, and use lower antibody concentrations (1:50-1:100)

  • Approach: Include additional negative controls such as secondary-only staining

• Reproducibility between lots:

  • Pitfall: Variation in performance between antibody lots, particularly with polyclonal antibodies

  • Solution: Validate each new lot against previous standards using consistent positive controls

  • Approach: Consider generating stable cell lines expressing tagged CDCA7 as consistent controls

• Post-translational modification detection:

  • Pitfall: Failure to detect modified forms of CDCA7, which undergoes phosphorylation and potentially other modifications

  • Solution: Use phosphatase treatments on control samples to confirm if bands of unexpected mobility represent phosphorylated forms

  • Approach: Consider using phospho-specific antibodies if studying specific CDCA7 modifications

By anticipating these common challenges and implementing the suggested strategies, researchers can significantly improve the reliability and reproducibility of their CDCA7 antibody-based experiments.

How are CDCA7 antibodies being used to study epigenetic regulation in development and disease?

CDCA7 antibodies are increasingly employed in cutting-edge epigenetic research, revealing the protein's critical roles in development and disease:

• Development and stem cell biology:

  • CDCA7 antibodies have helped identify the protein as an evolutionary conserved Notch target involved in hematopoietic stem cell (HSC) emergence

  • IF and ChIP studies show CDCA7 expression is highest in human hemogenic endothelial precursors (HEPs), with expression dependent on Notch signaling

  • Tracking CDCA7 expression during embryonic development reveals its role in critical developmental transitions, particularly in hematopoiesis

• ICF syndrome mechanisms:

  • Antibodies against wild-type and mutant CDCA7 have been crucial in understanding how ICF syndrome mutations affect protein function

  • Studies show that ICF3 mutations in the CRD disrupt CDCA7's ability to concentrate in heterochromatin foci during S-phase, without affecting its interaction with HELLS

  • This has led to a mechanistic understanding that CDCA7 controls DNA methylation specificity by recognizing non-B DNA structures formed in juxtacentromeric regions during replication

• Epigenome maintenance mechanisms:

  • CDCA7 antibodies have been instrumental in characterizing protein complexes involved in maintaining epigenetic marks during cell division

  • Recent findings show CDCA7 functions as a hemimethylated DNA adaptor for the nucleosome remodeler HELLS

  • The protein selectively binds hemimethylated DNA with strand-specific CpG methylation through its zinc-binding 4CXXC_R1 domain

• Cancer epigenetics:

  • CDCA7 overexpression in cancer correlates with altered DNA methylation patterns and transcriptional regulation

  • Antibody-based studies have linked CDCA7 to MYC-mediated transformation, providing insights into how cancer cells reprogram their epigenome

Recent studies using cryo-EM with antibody validation have revealed that CDCA7's C-terminal region contains a unique zinc-binding structure that recognizes CpG dyads in non-B DNA conformations . This structural insight explains how CDCA7 directs DNA methylation to specific genomic regions and why ICF syndrome mutations disrupt this function.

What novel techniques are being developed that incorporate CDCA7 antibodies?

Researchers are developing innovative techniques that leverage CDCA7 antibodies to advance our understanding of epigenetic regulation and disease mechanisms:

• Proximity-based protein interaction mapping:

  • CDCA7 antibodies are being utilized in proximity ligation assays (PLA) to visualize and quantify interactions with partners like HELLS in situ

  • BioID or APEX2-based proximity labeling with CDCA7 antibodies for validation is revealing novel interaction partners in different cellular compartments

  • These approaches can identify cell-type specific or cell-cycle dependent interactions that may be missed in traditional co-IP experiments

• Live-cell imaging techniques:

  • While not directly using antibodies, the validation of CDCA7 fluorescent protein fusions with antibodies enables live-cell tracking of CDCA7 dynamics

  • These approaches reveal CDCA7's real-time recruitment to newly synthesized DNA during replication

  • Time-lapse imaging combined with cell cycle markers provides insights into the temporal regulation of CDCA7 function

• Single-cell epigenomic profiling:

  • CDCA7 antibodies are being incorporated into CUT&Tag or CUT&RUN protocols for high-resolution mapping of CDCA7 binding sites

  • When combined with single-cell technologies, these approaches can reveal cell-to-cell heterogeneity in CDCA7 function

  • This is particularly relevant for understanding CDCA7's role in developmental transitions and cancer heterogeneity

• Therapeutic targeting validation:

  • As CDCA7 emerges as a potential therapeutic target in cancer, antibodies are essential for validating the efficacy of inhibitors

  • Techniques combining antibody-based detection with functional assays can assess how targeting CDCA7 affects downstream pathways

  • In pancreatic cancer, such approaches have demonstrated that targeting CDCA7 might increase gemcitabine sensitivity by inhibiting glycolysis

• Structural determination approaches:

  • Antibody-validated recombinant domains of CDCA7 have been crucial for structural studies

  • Recent work has revealed that the CDCA7 CRD adopts a unique zinc-binding structure for DNA recognition

  • These structural insights are enabling structure-based design of specific inhibitors with potential therapeutic applications

Emerging methodologies coupling CDCA7 antibodies with high-throughput sequencing, such as CUT&Tag-seq, are providing genome-wide views of CDCA7 binding patterns in different cell types and disease states. These approaches are particularly valuable for understanding how CDCA7 contributes to maintaining cell-type specific epigenetic landscapes and how its dysregulation contributes to disease development.

What are the key considerations for selecting and validating CDCA7 antibodies for specific research applications?

When selecting and validating CDCA7 antibodies for specific research applications, researchers should consider several critical factors:

• Research question alignment:

  • For isoform-specific studies, select antibodies raised against unique regions of particular isoforms

  • For interaction studies, ensure the antibody epitope doesn't overlap with known protein-interaction domains

  • For functional studies, consider antibodies that don't interfere with CDCA7's DNA binding or protein interaction capabilities

• Application-specific validation:

  • Each application (WB, IP, IF, IHC) requires specific validation approaches

  • Cross-application performance cannot be assumed; an antibody performing well in Western blot may not work for immunofluorescence

  • Use the recommended dilutions as starting points: WB (1:1000-1:5000), IP (0.5-4.0 μg), IHC (1:250-1:1000), and IF (1:10-1:100)

• Species consideration:

  • Verify reactivity with target species; common CDCA7 antibodies show reactivity with human, mouse, and rat samples

  • Be aware that epitope conservation may vary across species, potentially affecting antibody performance

• Experimental controls:

  • Implement genetic controls (CDCA7 knockout or knockdown)

  • Include peptide competition assays

  • Compare multiple antibodies targeting different epitopes

• Technical aspects:

  • Consider the advantages of monoclonal versus polyclonal antibodies based on research needs

  • For detecting post-translational modifications, select antibodies that are not affected by these modifications unless specifically studying them

  • Be aware of buffer compatibility issues that may affect antibody performance

The reproducibility challenges reported with some commercial CDCA7 antibodies highlight the importance of rigorous validation. Researchers should maintain detailed records of antibody performance across different lots and applications to ensure experimental reproducibility and reliable data interpretation.

What are the future directions for CDCA7 antibody applications in research and potential clinical settings?

The future of CDCA7 antibody applications spans both basic research advancements and translational clinical applications:

• Basic research advancements:

  • Development of conditional degron systems validated by CDCA7 antibodies to study acute protein depletion effects

  • Combination with CRISPR screens to identify synthetic lethal interactions in CDCA7-dependent pathways

  • Integration with spatial transcriptomics to correlate CDCA7 localization with regional gene expression patterns

  • Application in organoid models to study CDCA7's role in tissue development and disease progression

• Diagnostic applications:

  • Development of standardized IHC protocols for CDCA7 detection in cancer tissue samples

  • CDCA7 antibody-based assessment as a potential prognostic marker in pancreatic cancer and lymphomas

  • Correlation of CDCA7 expression patterns with treatment response and patient outcomes

  • Combination with other biomarkers to develop diagnostic panels for early cancer detection

• Therapeutic developments:

  • Validation of CDCA7-targeting therapeutics using antibody-based detection methods

  • Development of antibody-drug conjugates targeting CDCA7 in cancer cells with high surface expression

  • CDCA7 antibodies as tools for identifying patients likely to respond to epigenetic therapies

  • Monitoring treatment efficacy through changes in CDCA7 expression or localization patterns

• Technological innovations:

  • Development of highly specific monoclonal antibodies against different CDCA7 domains and isoforms

  • Creation of conformation-specific antibodies that recognize active versus inactive CDCA7

  • Integration with artificial intelligence algorithms to analyze CDCA7 expression patterns in large patient cohorts

  • Incorporation into microfluidic and single-cell analysis platforms for high-throughput screening

• Epigenetic medicine applications:

  • Use of CDCA7 antibodies to stratify ICF syndrome patients and guide personalized treatment approaches

  • Development of companion diagnostics for epigenetic therapies targeting CDCA7-dependent pathways

  • Monitoring epigenetic reprogramming during treatment through changes in CDCA7 localization patterns

As our understanding of CDCA7's roles in health and disease continues to expand, antibody-based research tools will remain essential for elucidating its functions and developing targeted interventions. The emerging connections between CDCA7 and cancer metabolism , DNA methylation regulation , and developmental processes suggest that CDCA7 antibodies will play increasingly important roles in both mechanistic studies and translational research.