CCDC89 Antibody

What is CCDC89 and what are its key characteristics?

CCDC89, also known as Bc8 orange-interacting protein (BOIP), is a protein-coding gene that produces a protein characterized by its coiled-coil domain structure. The human protein has a UniProt ID of Q8N998 and an Entrez Gene ID of 220388 . At the molecular level, it has 374 amino acid residues with a mass of approximately 43.8 kDa . The protein demonstrates dual localization in both nuclear and cytoplasmic compartments , suggesting potential roles in nucleocytoplasmic transport or compartment-specific functions. CCDC89 shows notable expression in specific tissues, particularly in the testis, nasopharynx, and fallopian tube , which may indicate specialized functions in reproductive and respiratory systems.

The protein's coiled-coil domains typically facilitate protein-protein interactions, suggesting CCDC89 may function in complex formation or as a structural scaffold. Sequence analysis reveals significant conservation across species, with human CCDC89 showing approximately 83% identity with mouse orthologs and 81% with rat orthologs in the immunogenic regions used for antibody production .

CCDC89 antibodies have been validated for multiple experimental applications:

• Western Blotting (WB): Effective for quantification and molecular weight confirmation of CCDC89, typically showing a band at approximately 43.8 kDa. Recommended dilutions range from 1:100-1:1000 .

• Immunohistochemistry (IHC): Useful for visualizing CCDC89 distribution in tissue sections, revealing both intensity and localization patterns. Typically performed at dilutions of 1:100-1:500 .

• Immunocytochemistry/Immunofluorescence (ICC/IF): Provides higher resolution visualization of subcellular localization, confirming CCDC89's distribution between nuclear and cytoplasmic compartments. Effective at dilutions of 1:50-1:200 .

• Flow Cytometry: Can be used to quantify CCDC89 expression levels across cell populations, particularly with conjugated antibodies .

• Immunoprecipitation (IP): Though not explicitly validated in all sources, the availability of affinity-purified antibodies suggests potential utility in co-immunoprecipitation studies for identifying interaction partners .

Each application requires specific optimization of conditions including antibody concentration, incubation parameters, and detection methods to maximize specific signal while minimizing background.

What are the optimal conditions for detecting CCDC89 in Western blot applications?

Successful Western blot detection of CCDC89 requires careful optimization of multiple parameters:

Sample Preparation:

• Use extraction buffers containing both non-ionic detergents (0.5-1% Triton X-100) and ionic detergents (0.1% SDS) to efficiently solubilize both nuclear and cytoplasmic CCDC89 .

• Include protease inhibitor cocktails to prevent degradation of the target protein.

• Consider preparing separate nuclear and cytoplasmic fractions to assess compartment-specific expression.

Electrophoresis and Transfer:

• 10-12% polyacrylamide gels provide optimal resolution for the 43.8 kDa CCDC89 protein.

• Transfer efficiency is critical; semi-dry transfer at 15V for 30-45 minutes or wet transfer at 100V for 1 hour is generally effective.

• Verify transfer using reversible total protein stains (Ponceau S) before immunodetection.

Antibody Incubation:

• Blocking with 5% non-fat dry milk in TBST is typically effective, though some antibodies may perform better with BSA-based blocking buffers.

• Primary antibody dilutions between 1:100-1:1000 in blocking buffer are recommended, with overnight incubation at 4°C often yielding optimal results .

• Secondary antibody selection should match the host species of the primary antibody (typically rabbit for most commercial CCDC89 antibodies) .

Detection and Troubleshooting:

• Enhanced chemiluminescence detection provides adequate sensitivity for most applications.

• If signal is weak, consider using tissues with known higher CCDC89 expression (testis, nasopharynx, fallopian tube) as positive controls .

• For problematic detection, epitope masking by post-translational modifications might be occurring; sample treatment with phosphatases may reveal masked epitopes.

How can researchers validate the specificity of CCDC89 antibodies?

Validating antibody specificity is crucial for generating reliable data. A comprehensive validation approach includes:

• Antibody Titration: Test dilutions from 1:50 to 1:500 to identify optimal signal-to-noise ratio using tissues or cells known to express CCDC89.

• Peptide Competition Assays: Pre-incubate the antibody with excess immunizing peptide (when available), which should abolish specific staining.

• Multiple Antibody Comparison: Compare staining patterns using antibodies targeting different CCDC89 epitopes; consistent patterns strongly support specificity.

• Correlation with mRNA Expression: Compare protein detection with CCDC89 mRNA expression data from the same tissues or cells.

• Genetic Controls: When possible, use tissues or cells with CCDC89 knockdown/knockout as negative controls.

• Western Blot Verification: Confirm that the antibody detects a band of the expected molecular weight (approximately 43.8 kDa) in the same samples used for other applications.

• Cross-reactivity Assessment: Test the antibody on samples from multiple species to confirm expected cross-reactivity patterns based on sequence homology (e.g., human, mouse with 83% homology, rat with 81% homology) .

Implementing these validation steps provides confidence in the specificity of experimental findings and facilitates accurate interpretation of results.

What are the recommended fixation and permeabilization methods for CCDC89 immunofluorescence?

Optimizing fixation and permeabilization is critical for accurate visualization of CCDC89's dual nuclear-cytoplasmic localization:

Fixation Options:

• 4% paraformaldehyde (15-20 minutes at room temperature) generally preserves both protein antigenicity and cellular architecture.

• Methanol fixation (100% methanol at -20°C for 10 minutes) often enhances nuclear protein detection and may be preferable for nuclear CCDC89 visualization.

• A combination approach of brief PFA fixation followed by methanol post-fixation may provide optimal results for dual-localized proteins like CCDC89.

Permeabilization Methods:

• 0.1-0.3% Triton X-100 in PBS (10 minutes) effectively permeabilizes both plasma and nuclear membranes.

• For antibodies demonstrating weaker nuclear signal, increasing permeabilization time or detergent concentration may enhance nuclear epitope accessibility.

Blocking and Antibody Incubation:

• Block with 5-10% normal serum from the secondary antibody species, with 1% BSA addition to reduce non-specific binding.

• Primary antibody dilutions between 1:50-1:200 are typically effective for immunofluorescence .

• Overnight incubation at 4°C often yields superior results compared to shorter incubations at room temperature.

Controls and Validation:

• Include nuclear and cytoplasmic markers (e.g., lamin A/C for nuclear, GAPDH for cytoplasmic) to confirm proper compartment preservation.

• Compare staining patterns across multiple fixation methods to ensure complete epitope accessibility.

• After optimization, validate the protocol across multiple cell types relevant to CCDC89 biology.

How can researchers effectively study CCDC89 in specific subcellular compartments?

Given CCDC89's dual localization, compartment-specific analysis requires specialized approaches:

Biochemical Fractionation:

• Optimize nuclear-cytoplasmic separation protocols using hypotonic lysis followed by differential centrifugation.

• Verify fraction purity using compartment-specific markers (lamin A/C for nuclear, GAPDH for cytoplasmic fractions).

• Consider additional purification to separate nucleoplasmic from chromatin-bound fractions for more detailed analysis.

Imaging Approaches:

• Perform high-resolution confocal microscopy with co-staining for compartment markers.

• Super-resolution techniques (STORM, STED) can provide nanoscale localization precision for detecting potential subcompartment associations.

Experimental Manipulation of Localization:

• Generate constructs with modified or additional nuclear localization signals (NLS) or nuclear export signals (NES) to enforce compartment-specific accumulation.

• Implement the anchor-away technique to conditionally remove CCDC89 from specific compartments.

Compartment-Specific Interaction Studies:

• Use proximity labeling approaches (BioID, APEX) with CCDC89 fusions to identify compartment-specific interaction partners.

• Perform co-immunoprecipitation from purified subcellular fractions to identify compartment-specific binding partners.

Dynamic Analysis:

• Consider that localization may change with cell cycle stage, differentiation state, or in response to cellular signals.

• Live-cell imaging with fluorescently tagged CCDC89 can track protein movement between compartments in response to stimuli.

How should researchers troubleshoot weak or absent CCDC89 signal?

When encountering detection problems, implement this systematic troubleshooting approach:

For application-specific troubleshooting:

• Western blot: Verify transfer efficiency with reversible stains; consider enhanced chemiluminescence substrates for improved sensitivity.

• IHC/ICC: Optimize antigen retrieval methods; test multiple fixation protocols to preserve epitope accessibility.

• IP applications: Increase antibody amount (3-5μg per 500μg total protein); extend incubation time.

What controls should be included in CCDC89 antibody experiments?

Implementing appropriate controls is essential for reliable interpretation:

Positive Controls:

• Include tissues with known CCDC89 expression (testis, nasopharynx, fallopian tube) .

• Consider overexpression systems as strong positive controls for antibody validation.

Negative Controls:

• No-primary-antibody controls to assess secondary antibody specificity.

• Isotype controls (non-specific antibodies of the same isotype) to distinguish specific binding.

• When available, CCDC89 knockdown/knockout samples provide definitive negative controls.

Specificity Controls:

• Peptide competition/neutralization controls by pre-incubating antibody with immunizing peptide.

• Testing multiple antibodies targeting different CCDC89 epitopes for consistent patterns.

Technical Controls:

• For multiplexed experiments, single-color controls to assess spectral overlap.

• For quantitative applications, standard curves with recombinant protein (when available).

These controls should be systematically implemented across experimental replicates to ensure data reliability and facilitate accurate interpretation of results.

How can CCDC89 antibodies be used to investigate protein-protein interactions?

Investigating CCDC89 interactions requires strategic application of antibody-based techniques:

Co-immunoprecipitation (Co-IP):

• Use CCDC89 antibodies to pull down the protein complex, followed by immunoblotting for suspected interaction partners.

• Optimize lysis conditions to preserve interactions (mild detergents, physiological salt concentrations).

• Consider crosslinking approaches to stabilize transient interactions before cell lysis.

• Include appropriate controls: IgG control IPs, reverse IPs (immunoprecipitating suspected partners).

Proximity Labeling:

• Generate BioID or APEX2 fusion constructs with CCDC89 to identify proteins in close proximity.

• Use antibodies to verify candidate interactions identified through mass spectrometry.

Fluorescence-based Interaction Analysis:

• Implement Förster Resonance Energy Transfer (FRET) or Bimolecular Fluorescence Complementation (BiFC) using antibody-validated constructs.

• Use immunofluorescence to confirm co-localization of CCDC89 with potential interaction partners.

Interaction Domain Mapping:

• Generate truncation or point mutants of CCDC89 to determine which regions mediate specific interactions.

• Use antibodies recognizing different domains to confirm expression of truncated constructs.

Given CCDC89's dual subcellular localization, interaction studies should examine nuclear and cytoplasmic fractions separately to identify compartment-specific binding partners that might suggest distinct functions .

What are the considerations for using CCDC89 antibodies in multiplex immunofluorescence?

Implementing CCDC89 antibodies in multiplex studies requires careful planning:

Antibody Compatibility:

• Select primary antibodies from different host species to enable species-specific secondary detection.

• If same-species antibodies are necessary, consider direct conjugation of primaries or sequential detection protocols.

Fixation and Antigen Retrieval:

• Identify fixation protocols compatible with all target epitopes in the multiplex panel.

• If antigen retrieval is needed, ensure methods preserve all target epitopes simultaneously.

Signal Optimization:

• Address CCDC89's dual localization by selecting appropriate fluorophores with sufficient brightness.

• Balance signal intensity across all markers in the panel by optimizing antibody concentrations.

Controls for Multiplex Systems:

• Implement single-antibody staining controls for each component of the multiplex panel.

• Include fluorescence-minus-one controls to assess bleed-through and cross-reactivity.

Image Acquisition and Analysis:

• Configure acquisition settings to balance detection of all signals while avoiding spectral bleed-through.

• Consider using spectral unmixing algorithms for closely overlapping fluorophores.

• Implement quantitative analysis strategies that account for CCDC89's distribution across multiple compartments.

By addressing these considerations, researchers can effectively incorporate CCDC89 antibodies into complex multiplex studies while maintaining data reliability and interpretability.

What are the key considerations for evaluating CCDC89 expression changes?

Accurately assessing CCDC89 expression changes requires attention to several factors:

Experimental Design:

• Include appropriate controls: vehicle-treated samples for chemical treatments, empty vector controls for overexpression studies.

• Implement time-course analyses to capture transient expression changes that might be missed at single timepoints.

Quantification Methods:

• Normalize to appropriate loading controls: housekeeping proteins for modest changes, total protein staining for larger changes.

• For CCDC89 specifically, consider separate analysis of nuclear and cytoplasmic fractions, as distribution shifts between compartments may occur without total protein level changes .

Complementary Approaches:

• Combine protein-level analysis (Western blot, immunostaining) with mRNA assessment (qRT-PCR) to determine whether expression changes occur at transcriptional or post-transcriptional levels.

• Design primers targeting regions common to all known CCDC89 splice variants or use variant-specific primers to detect isoform-specific regulation.

Interpretation Considerations:

• Consider that apparent expression changes might reflect altered antibody epitope accessibility rather than true expression differences.

• Confirm results with multiple antibodies targeting distinct CCDC89 epitopes.

• Establish biological significance thresholds—statistical significance may not equate to biological relevance.

What techniques can help distinguish between potential CCDC89 isoforms?

Distinguishing CCDC89 isoforms requires a strategic combination of approaches:

Bioinformatic Analysis:

• Utilize genomic and transcriptomic databases to identify predicted isoforms, their theoretical molecular weights, and unique sequences.

Western Blot Optimization:

• Implement high-resolution SDS-PAGE (8-10% gels run at lower voltage) to separate closely sized isoforms.

• Use antibodies targeting common regions to detect all isoforms simultaneously.

• Apply domain-specific antibodies that may recognize distinct subsets of isoforms.

2D Electrophoresis:

• Combine isoelectric focusing with SDS-PAGE to resolve isoforms with similar molecular weights but different charges.

Transcript Analysis:

• Employ RT-PCR with isoform-specific primers spanning unique exon junctions.

• Implement qRT-PCR to quantify relative isoform expression across tissues or experimental conditions.

Mass Spectrometry:

• For definitive identification, perform immunoprecipitation followed by mass spectrometry to identify peptides unique to specific isoforms.

Controls and Validation:

• Generate positive controls by overexpressing individual CCDC89 isoforms with epitope tags to serve as size markers and specificity controls.

• Consider that isoform expression may vary by tissue type, developmental stage, or cellular condition.

By integrating these approaches, researchers can effectively distinguish between CCDC89 isoforms and investigate their potentially distinct functions.