CCDC97 Antibody

What is CCDC97 and what are its primary cellular functions?

CCDC97 is a 39 kDa protein containing coiled-coil domains, identified by the UniProt accession number Q96F63 and gene ID 90324 . It plays a significant role in pre-mRNA splicing through its association with the splicing factor SF3B complex, which is involved in branch-site recognition . The protein is predominantly expressed in human tissues, with recent research demonstrating elevated expression in hepatocellular carcinoma (HCC) tissues compared to normal liver tissues . Functionally, CCDC97 appears to influence cellular processes including migration, invasion, and proliferation, as demonstrated by knockdown studies in HCC cell lines . Its activity in the spliceosome pathway may contribute to its role in carcinogenesis, as this pathway is significantly active in various tumors .

What types of CCDC97 antibodies are currently available for research applications?

Most commercially available CCDC97 antibodies are rabbit polyclonal antibodies that target different epitopes of the human CCDC97 protein. These include:

Several conjugated variants are also available, including HRP-conjugated, FITC-conjugated, and biotin-conjugated antibodies for specialized applications . The majority of these antibodies have been validated against human samples, with some showing cross-reactivity with cow, horse, and pig tissues .

How should optimal dilutions be determined for different experimental applications?

Determining optimal dilutions for CCDC97 antibodies varies by application:

Researchers should always perform titration experiments with positive controls such as Jurkat cells to determine the optimal concentration for their specific experimental conditions . The signal-to-noise ratio should be evaluated across a dilution series, with the optimal dilution providing clear specific bands or staining with minimal background.

What validation strategies should be employed to confirm CCDC97 antibody specificity?

A multi-faceted validation approach is essential for confirming CCDC97 antibody specificity:

• Positive and negative cell lines: Use established positive controls such as Jurkat, HeLa, and HEK-293T cell lines for antibody validation . Include cell lines with low or no CCDC97 expression as negative controls.

• Genetic approaches: Employ siRNA knockdown or CRISPR-Cas9 knockout of CCDC97 to verify the specificity of antibody binding. The absence or significant reduction of signal in knockdown/knockout samples compared to controls provides strong evidence of specificity .

• Multiple antibody validation: Use multiple antibodies targeting different epitopes of CCDC97 and compare their staining patterns. Convergent results from different antibodies increase confidence in specificity .

• Molecular weight verification: Confirm that the detected band in Western blot corresponds to the predicted molecular weight of CCDC97 (39 kDa) .

• Recombinant protein controls: Use purified recombinant CCDC97 protein as a positive control and for pre-absorption tests to confirm specificity.

The enhanced validation approaches mentioned for some antibodies include recombinant expression validation and orthogonal RNA sequencing, which provide additional confidence in antibody specificity .

How can researchers optimize immunohistochemistry protocols for CCDC97 detection in tissue samples?

Optimizing IHC protocols for CCDC97 requires attention to several critical parameters:

• Tissue fixation: Standard 10% neutral-buffered formalin fixation for 24-48 hours is typically suitable, but optimization may be required for specific tissues.

• Antigen retrieval: Heat-induced epitope retrieval using citrate buffer (pH 6.0) or EDTA buffer (pH 9.0) should be tested to determine which provides optimal staining. Perform retrieval for 15-20 minutes at 95-100°C.

• Blocking procedure: Use 5-10% normal serum (from the same species as the secondary antibody) with 1% BSA in PBS for 1 hour at room temperature to minimize non-specific binding.

• Primary antibody incubation: Apply CCDC97 antibody at the recommended dilution (1:100-1:1000) and incubate overnight at 4°C in a humidified chamber.

• Detection system: Use a sensitive detection system appropriate for the application (HRP-polymer or fluorescent secondary antibodies).

• Counterstaining: Hematoxylin counterstaining (for brightfield) or DAPI (for fluorescence) helps visualize tissue architecture.

• Controls: Include positive control tissues known to express CCDC97 and negative controls (primary antibody omitted or isotype control) in each experiment.

Researchers should evaluate both the intensity and pattern of staining, as CCDC97 may exhibit both nuclear and cytoplasmic localization depending on the tissue context and its functional state in the splicing machinery.

What are the critical parameters for successful Western blot detection of CCDC97?

Several critical parameters influence Western blot success for CCDC97 detection:

• Sample preparation: Use RIPA buffer supplemented with protease inhibitors for efficient extraction. For nuclear proteins, consider using specialized nuclear extraction protocols.

• Protein loading: Load 50 μg of total protein per lane, as demonstrated in published Western blots . Adjust as needed based on CCDC97 expression levels in your samples.

• Gel percentage: Use 10-12% SDS-PAGE gels for optimal separation around the 39 kDa range.

• Transfer conditions: Semi-dry transfer at 15V for 30-45 minutes or wet transfer at 100V for 1 hour at 4°C using PVDF membranes (0.45 μm pore size).

• Blocking: Block with 5% non-fat dry milk or BSA in TBST for 1 hour at room temperature.

• Antibody incubation: Apply primary antibody at the recommended dilution (0.1 μg/mL or 1:500-1:3000) in blocking buffer overnight at 4°C.

• Detection: Use ECL (enhanced chemiluminescence) for detection, with exposure times starting at 30 seconds and adjusting as needed.

• Expected results: Anticipate a band at approximately 39 kDa, which is the predicted molecular weight of CCDC97 .

For challenging samples, consider enrichment techniques such as immunoprecipitation before Western blotting, which has been successfully employed with CCDC97 antibodies in HEK-293T cell lysates .

What are common challenges in CCDC97 detection and how can they be addressed?

Researchers frequently encounter several challenges when working with CCDC97 antibodies:

• Weak or absent signal:

  • Increase antibody concentration or incubation time

  • Optimize antigen retrieval conditions (for IHC)

  • Use more sensitive detection systems (e.g., amplification systems)

  • Confirm CCDC97 expression in your sample type through transcriptomic data

  • Ensure proper sample handling to prevent protein degradation

• High background:

  • Increase blocking time or concentration

  • Reduce primary antibody concentration

  • Add 0.1-0.3% Triton X-100 to washing buffer to reduce non-specific binding

  • Increase washing steps duration and number

  • Use more dilute secondary antibody

• Multiple bands in Western blot:

  • Optimize sample preparation to reduce protein degradation

  • Use freshly prepared samples and buffers

  • Run gradient gels for better separation

  • Consider the possibility of post-translational modifications or splice variants

  • Validate with siRNA knockdown to identify the specific CCDC97 band

• Inconsistent results across experiments:

  • Standardize protocols with detailed SOPs

  • Use the same lot of antibody when possible

  • Include consistent positive controls in each experiment

  • Implement quantitative measures for normalization

For quantitative analysis of CCDC97 expression, normalize Western blot data to appropriate loading controls and IHC/ICC staining to reference markers using digital image analysis software.

How should researchers interpret contradictory results between different CCDC97 antibodies or detection methods?

When faced with contradictory results, implement the following analytical approach:

• Epitope mapping: Consider the epitope location of each antibody. Different antibodies targeting distinct regions (N-terminal, central, or C-terminal) may yield different results if:

  • Protein interactions mask certain epitopes

  • Post-translational modifications affect antibody binding

  • Alternative splicing creates isoforms lacking specific epitopes

• Method-specific limitations: Recognize that each detection method has inherent limitations:

  • Western blot evaluates denatured protein, potentially missing conformational epitopes

  • IHC may be affected by fixation-induced epitope masking

  • ICC reflects in vitro conditions that may not represent in vivo expression

• Biological variability: Consider that CCDC97 expression and localization may vary based on:

  • Cell cycle stage

  • Cellular stress conditions

  • Differentiation status

  • Disease context (particularly in cancer)

• Validation hierarchy: Establish a hierarchical approach to resolve contradictions:

  • Genetic manipulation (knockdown/knockout) provides the strongest validation

  • Orthogonal methods (protein vs. RNA detection)

  • Multiple antibodies targeting different epitopes

  • Correlation with functional assays

What experimental controls are essential when investigating CCDC97 function in cellular processes?

A comprehensive control strategy is vital for functional CCDC97 studies:

• Expression controls:

  • Positive cellular controls: Jurkat, HeLa, and HEK-293T cells have confirmed CCDC97 expression

  • Negative controls: Cells with naturally low CCDC97 expression or CCDC97-knockout cells

  • Loading controls: β-actin, GAPDH, or tubulin for protein normalization

• Genetic manipulation controls:

  • siRNA controls: Non-targeting siRNA for knockdown experiments

  • Multiple siRNAs targeting different regions of CCDC97 to confirm specificity

  • Rescue experiments: Re-expression of siRNA-resistant CCDC97 to restore function

• Localization studies:

  • Co-staining with markers for subcellular compartments (nuclear, nuclear speckles, cytoplasmic)

  • Primary antibody omission and isotype controls for immunostaining

  • GFP-tagged CCDC97 for live-cell imaging validation

• Functional assays:

  • Time-course experiments to establish causality

  • Dose-response relationships in overexpression/knockdown studies

  • Comparison with known splicing factors as reference points

• Disease model controls:

  • Matched normal-tumor pairs when studying cancer contexts

  • Stage-specific samples to track progression-related changes

  • Patient-derived materials to validate cell line findings

Researchers investigating CCDC97's role in splicing should particularly include controls that distinguish direct from indirect effects, such as RNA immunoprecipitation controls and comparisons with established splicing factors.

How can CCDC97 antibodies be utilized to investigate pre-mRNA splicing mechanisms?

CCDC97 antibodies offer several approaches to investigate splicing mechanisms:

• Co-immunoprecipitation (Co-IP): Use CCDC97 antibodies for Co-IP to identify interacting partners within the spliceosome complex, particularly components of the SF3B complex with which CCDC97 is associated . This approach can reveal:

  • Direct protein-protein interactions

  • Complex composition variations across cell types

  • Changes in interactions under different cellular conditions

• Chromatin Immunoprecipitation (ChIP): Employ ChIP to investigate CCDC97 association with chromatin and nascent RNA transcripts, which can provide insights into:

  • Co-transcriptional versus post-transcriptional splicing roles

  • Genomic binding sites and motif preferences

  • Integration with transcriptional regulation

• RNA Immunoprecipitation (RIP): Use RIP to identify RNA targets of CCDC97:

  • Map binding sites on pre-mRNAs

  • Identify RNA sequence or structural preferences

  • Correlate binding with alternative splicing outcomes

• Immunofluorescence microscopy: Visualize CCDC97 localization in nuclear speckles and its co-localization with other splicing factors:

  • Track dynamic changes during the cell cycle

  • Observe responses to splicing inhibitors

  • Analyze redistribution following cellular stress

• Proximity ligation assays (PLA): Detect in situ interactions between CCDC97 and other splicing factors with single-molecule resolution.

These approaches, combined with functional splicing assays following CCDC97 manipulation, can elucidate its specific role in the splicing machinery and potentially identify unique regulatory functions beyond the core spliceosome activity.

What is the significance of CCDC97 in cancer research, particularly hepatocellular carcinoma?

Recent research has revealed CCDC97 as a potentially important factor in cancer biology, with particular relevance to hepatocellular carcinoma (HCC):

• Expression pattern: CCDC97 shows elevated expression in HCC patients and HCC cell lines compared to normal controls, suggesting a potential role in carcinogenesis .

• Clinical correlations: CCDC97 expression is closely associated with pathological features and prognosis in HCC patients, positioning it as a novel prognostic biomarker .

• Functional impact: Experimental knockdown of CCDC97 in vitro suppressed cancer cell migration, invasion, and proliferation, indicating a potential oncogenic function .

• Pathway involvement: CCDC97 is linked to the spliceosome pathway, which is significantly active in tumors and may contribute to carcinogenesis through altered splicing of cancer-related genes .

• Immune microenvironment: CCDC97 is also highly expressed in various immune cells and is associated with the tumor immune microenvironment, suggesting a role in tumor-immune interactions .

These findings collectively position CCDC97 as a promising target for further investigation in HCC and potentially other cancers. Researchers can use CCDC97 antibodies to:

• Evaluate expression in patient samples for correlation with clinical outcomes

• Investigate altered splicing patterns in CCDC97-high versus CCDC97-low tumors

• Explore the impact of CCDC97 on immune cell infiltration and function within tumors

• Develop and test therapeutic strategies targeting CCDC97 or its downstream effectors

What methodological approaches should be considered when investigating CCDC97 as a potential therapeutic target?

Investigating CCDC97 as a therapeutic target requires a multi-faceted methodological approach:

• Target validation strategies:

  • Genetic manipulation: Use siRNA, shRNA, or CRISPR-Cas9 to modulate CCDC97 expression and assess phenotypic consequences

  • Dose-response relationships: Establish how varying levels of CCDC97 suppression correlate with biological effects

  • Tissue specificity: Determine if CCDC97 dependency varies across tissue types or disease contexts

  • In vivo validation: Confirm in vitro findings using animal models with CCDC97 manipulation

• Mechanism of action studies:

  • Splicing alterations: Identify specific splice variants affected by CCDC97 modulation using RNA-seq and alternative splicing analysis

  • Signaling pathway integration: Map how CCDC97-mediated splicing changes affect oncogenic signaling networks

  • Immune modulation: Characterize changes in immune cell populations and function following CCDC97 manipulation

• Drug development approaches:

  • Small molecule screening: Identify compounds that disrupt CCDC97 interactions with the spliceosome

  • Protein-protein interaction inhibitors: Design molecules targeting specific interfaces within CCDC97 complexes

  • Degrader development: Consider PROTAC (Proteolysis Targeting Chimera) approaches to induce CCDC97 degradation

  • Antisense oligonucleotides: Target CCDC97 mRNA directly

• Biomarker development:

  • Expression analysis: Optimize IHC protocols for patient stratification based on CCDC97 expression

  • Functional assays: Develop tests to assess CCDC97-dependent splicing as a pharmacodynamic marker

  • Resistance mechanisms: Identify potential mechanisms of resistance to CCDC97-targeted therapies

• Combination strategies:

  • Synergy testing: Evaluate combinations with established therapies (chemotherapy, immunotherapy)

  • Synthetic lethality: Identify genetic contexts that enhance sensitivity to CCDC97 inhibition

When designing these studies, researchers should consider both on-target and off-target effects, given the fundamental role of splicing in cellular processes, and develop appropriate safety assessment strategies alongside efficacy evaluations.

How might novel antibody technologies advance CCDC97 research beyond current limitations?

The advancement of antibody technologies presents new opportunities for CCDC97 research:

• Single-domain antibodies: Nanobodies and single-chain variable fragments (scFvs) offer superior penetration into subcellular compartments and may provide better access to CCDC97 within the dense nuclear splicing machinery.

• Intrabodies: Genetically encoded antibody fragments that function inside cells could allow real-time visualization of CCDC97 dynamics during splicing events.

• Proximity-dependent labeling: Antibody-enzyme fusions (APEX2, BioID) could map the proximal proteome of CCDC97 in different cellular contexts.

• Degradation-inducing antibodies: Proteolysis-targeting chimeric antibodies could achieve acute depletion of CCDC97 to study immediate functional consequences.

• Bispecific antibodies: Dual-targeting antibodies could simultaneously detect CCDC97 and interacting partners to study complex formation in situ.

• Recombinant antibody engineering: The development of highly specific recombinant antibodies through phage display or yeast display technologies, as recently demonstrated with RFdiffusion for de novo antibody design , could overcome current limitations in CCDC97 antibody specificity and versatility.

These emerging technologies could address current limitations in studying transient CCDC97 interactions, capturing dynamic changes during splicing, and distinguishing between different functional pools of CCDC97 within cells.