CDCA7L Antibody

What is CDCA7L and what cellular localization patterns should researchers expect when using CDCA7L antibodies?

CDCA7L belongs to the JPO protein family and serves as a transcriptional regulator that participates in c-Myc-mediated cell transformation. When using immunofluorescence or immunohistochemistry techniques, researchers should expect to observe both nuclear and cytoplasmic localization patterns, with translocation dynamics dependent on cellular conditions.

According to immunofluorescence data from multiple antibody suppliers, CDCA7L predominantly localizes to the nucleus under standard culture conditions, with some cytoplasmic expression also detectable . Importantly, CDCA7L has been documented to translocate from the cytoplasm to the nucleus under dexamethasone induction, which researchers should consider when designing stimulation experiments .

When optimizing staining protocols, researchers should note that subcellular localization can vary based on:

• Cell type (epithelial vs. hematopoietic cells)

• Cell cycle phase (differential nuclear localization)

• Treatment conditions (particularly those affecting the Myc pathway)

What are the typical molecular weights observed for CDCA7L in Western blot applications?

When performing Western blot analysis for CDCA7L, researchers should expect to observe a primary band at approximately 52 kDa, which corresponds to the canonical isoform of the protein.

Based on validation data from commercial antibodies:

• The calculated molecular weight of CDCA7L is 52 kDa (454 amino acids)

• The observed molecular weight in Western blots consistently appears at 52 kDa across multiple cell lines including A549, HEK-293T, HeLa, K-562, and MCF-7 cells

Researchers should be aware that CDCA7L has 4 isoforms produced by alternative splicing, which may result in secondary bands in some tissue types . When troubleshooting unexpected banding patterns, consider:

• Sample preparation method (harsh lysis buffers may affect detection)

• Presence of post-translational modifications

• Cell-type specific expression of alternative isoforms

Which cell lines are most suitable for CDCA7L antibody validation?

Based on validation data from multiple commercial antibodies, the following cell lines consistently show detectable CDCA7L expression and are recommended for antibody validation:

For negative controls, researchers should consider using cell lines with CDCA7L knockdown via siRNA or CRISPR, as specific CDCA7L-negative cell lines are not well documented in the literature.

How can CDCA7L antibodies be used to study its interaction with c-Myc in transformation assays?

CDCA7L has been identified as a target gene of c-Myc and plays a role in c-Myc-mediated transformation. To study this relationship, researchers can employ co-immunoprecipitation (Co-IP) assays with CDCA7L antibodies.

Based on published methodologies:

• Co-immunoprecipitation protocol:

  • Prepare cell lysates in Triton X-100 lysis buffer (50 mM HEPES, pH 7.4, 250 mM NaCl, 0.1% Triton X-100, 10% glycerol, plus protease and phosphatase inhibitors)

  • Immunoprecipitate with anti-CDCA7L antibody conjugated to agarose beads

  • Incubate overnight at 4°C

  • Wash beads 5 times with lysis buffer

  • Elute proteins with LDS sample buffer and heat to 70°C for 10 min

  • Analyze by Western blot for co-precipitated c-Myc

• Transformation assay design:

  • Transfect cells with CDCA7L-expressing vectors and c-Myc constructs

  • Evaluate colony formation capacity in soft agar

  • Confirm CDCA7L and c-Myc expression levels by Western blot using validated antibodies

  • Compare transformation efficiency with appropriate controls (empty vector, c-Myc only, CDCA7L only)

Research has shown that CDCA7L can complement c-Myc transformation-defective mutant W135E and potentiate Myc-mediated transformation . Understanding this interaction is critical for cancer research focused on Myc-driven oncogenesis.

What is the relationship between CDCA7L and CDCA7, and how can researchers avoid antibody cross-reactivity?

CDCA7L (cell division cycle associated 7-like) and CDCA7 (cell division cycle associated 7) are related but distinct proteins with overlapping functions in cell cycle regulation and Myc-mediated processes. Researchers must carefully select antibodies to avoid cross-reactivity between these proteins.

Key differences between CDCA7 and CDCA7L:

• CDCA7 is 371 amino acids with a molecular weight of 42.6 kDa

• CDCA7L is 454 amino acids with a molecular weight of 52 kDa

• CDCA7 participates in MYC-mediated cell transformation and apoptosis; it induces anchorage-independent growth and clonogenicity in lymphoblastoid cells

• CDCA7L is also involved in MYC-mediated transformation but has distinct regulation patterns

To ensure specificity:

• Validate antibodies using cells with CDCA7 or CDCA7L knockdown

• Confirm antibody specificity using recombinant protein arrays where available

• Check epitope sequences for uniqueness between the two proteins

• Consider using isoform-specific antibodies when available

The Prestige Antibodies from Sigma-Aldrich are tested on protein arrays of 364 human recombinant protein fragments, which can help confirm specificity against related proteins .

How do CDCA7L antibodies perform in glioma research applications?

CDCA7L has been identified as a promoter of glioma proliferation by targeting cyclin D1 (CCND1) and predicts poor prognosis in glioma patients. Researchers studying CDCA7L in glioma should consider the following evidence-based approaches:

What are the optimal protocols for using CDCA7L antibodies in different applications?

Based on validated commercial antibodies, researchers should consider the following application-specific recommendations:

Western Blot Protocol:

• Recommended dilutions: 1:500-1:2000 or 1:1000-1:5000 depending on antibody

• Detection has been validated in multiple cell lines: A549, HEK-293T, HeLa, K-562, MCF-7, and Raji cells

• Expected molecular weight: 52 kDa

• Standard SDS-PAGE and transfer to PVDF membrane is suitable for detection

Immunohistochemistry Protocol:

• Recommended dilutions: 1:250-1:1000

• Antigen retrieval: Use TE buffer pH 9.0 (optimal) or citrate buffer pH 6.0 (alternative)

• Positive control tissues: Human colon cancer tissue has been validated

• Use appropriate blocking (5% normal serum or BSA) to reduce background

Immunofluorescence Protocol:

• Recommended dilutions: 1:10-1:100

• Validated cell lines include HepG2

• Use 4% paraformaldehyde fixation followed by permeabilization with 0.1% Triton X-100

• Counter-stain nuclei with DAPI to confirm nuclear localization pattern

Flow Cytometry Protocol:

• Recommended concentration: 0.25 μg per 10^6 cells in 100 μl suspension

• Validated in A431 cells for intracellular staining

• Use appropriate fixation (4% paraformaldehyde) and permeabilization (0.1% saponin)

What troubleshooting strategies should be employed when CDCA7L antibodies yield suboptimal results?

When working with CDCA7L antibodies, researchers may encounter various technical challenges. Here are evidence-based troubleshooting approaches for common issues:

For weak or absent Western blot signals:

• Optimize protein extraction: Use RIPA or Triton X-100 lysis buffers (50 mM HEPES, pH 7.4, 250 mM NaCl, 0.1% Triton X-100, 10% glycerol) with protease inhibitors

• Increase antibody concentration or extend incubation time

• Confirm protein expression in your cell line (CDCA7L is most highly expressed in thymus and small intestine)

• Consider using fluorescent secondary antibodies (IR700 anti-mouse or IR800 anti-rabbit) for enhanced sensitivity

For high background in immunohistochemistry:

• Optimize blocking (5% skim milk for 30 minutes is effective for some CDCA7 antibodies)

• Increase washing steps (5 washes with appropriate buffer)

• Dilute primary antibody further

• Test alternative antigen retrieval methods (TE buffer pH 9.0 vs. citrate buffer pH 6.0)

For immunoprecipitation optimization:

• Use 10 μL of antibody-conjugated agarose beads per immunoprecipitation

• Incubate overnight at 4°C for maximum binding

• Perform extensive washing (5 times with lysis buffer)

• Elute with appropriate buffer (200 μl of LDS sample buffer, heated to 70°C for 10 min)

How can post-translational modifications affect CDCA7L antibody recognition?

Post-translational modifications can significantly impact antibody recognition of CDCA7L. While specific CDCA7L modification data is limited, research on the related protein CDCA7 provides insights relevant to researchers working with CDCA7L antibodies:

• Phosphorylation:

  • CDCA7 is phosphorylated by AKT at threonine 163, which promotes binding to 14-3-3 proteins and affects subcellular localization

  • Researchers should consider that phosphorylation-specific antibodies may be needed to detect specific activation states of CDCA7L

  • Phosphorylation can alter epitope accessibility, potentially reducing antibody binding

• Epitope masking by protein interactions:

  • CDCA7 interaction with 14-3-3 proteins can mask epitopes and affect antibody recognition

  • For detecting total CDCA7L, choose antibodies targeting regions unlikely to be affected by protein-protein interactions

• Sample preparation considerations:

  • Include appropriate phosphatase inhibitors in lysis buffers to preserve phosphorylation states

  • For detecting specific modifications, consider using peptide competition assays to validate specificity, as demonstrated with CDCA7 (using peptides CDSKSPRRRTFPG vs. phosphorylated CDSKSPRRR(p)TFPG)

  • When studying CDCA7L translocation, cellular fractionation protocols should be optimized to preserve native protein modifications

• Cross-reactivity testing:

  • Verify antibody specificity against modified and unmodified forms of the protein when studying post-translational modifications

  • Consider using in vitro modification systems to generate control samples with defined modification states

What emerging applications of CDCA7L antibodies show promise in cancer research?

Based on current research findings, several promising directions for CDCA7L antibody applications in cancer research are emerging:

• Prognostic biomarker development:

  • CDCA7L expression levels are associated with poor prognosis in glioma patients

  • Researchers could develop standardized immunohistochemistry protocols using validated CDCA7L antibodies for prognostic assessment

  • Multi-marker panels incorporating CDCA7L staining might improve prognostic accuracy

• Therapeutic target validation:

  • CDCA7L promotes glioma cell growth through regulation of CCND1

  • Antibodies can be used to validate target engagement in preclinical studies of CDCA7L inhibitors

  • Immunoprecipitation using CDCA7L antibodies could help identify binding partners for therapeutic targeting

• c-Myc pathway analysis:

  • CDCA7L complements c-Myc transformation-defective mutants

  • Using CDCA7L antibodies in ChIP-seq experiments could identify downstream targets

  • Multiplexed immunofluorescence with c-Myc and CDCA7L antibodies could reveal co-localization in different tumor types

• Cell cycle regulation studies:

  • CDCA7L knockdown significantly inhibits proliferation and colony formation of U87 cells by blocking cell cycle progression in the G0/G1 phase

  • Flow cytometry protocols using CDCA7L antibodies combined with cell cycle markers could further elucidate its role in cell cycle control

These emerging applications highlight the importance of developing and validating highly specific CDCA7L antibodies for cancer research applications.