CCP110 Antibody

What is CCP110 and what cellular functions does it regulate?

CCP110, also known as centrosomal protein of 110 kDa or CP110, is a protein encoded by the CCP110 gene in humans. It functions as a centrosomal protein required for the centrosome to function as a microtubule organizing center and plays an essential role in centrosome duplication. CCP110 serves as a key negative regulator of ciliogenesis in collaboration with CEP97 by capping the mother centriole, thereby preventing cilia formation . It is also involved in promoting ciliogenesis and may play a role in the assembly of the mother centriole subdistal appendages, affecting the fusion of recycling endosomes to basal bodies during cilia formation . Additionally, CCP110 is required for correct spindle formation and has a role in regulating cytokinesis and genome stability via cooperation with calmodulin (CaM) and centrin (CETN2) .

What types of CCP110 antibodies are available for research applications?

Several types of CCP110 antibodies are available for research applications:

Each antibody has been validated for specific applications, and researchers should select the appropriate antibody based on their experimental requirements .

How does CCP110 interact with other centrosomal proteins?

CCP110 interacts with multiple proteins to regulate centrosome and cilia functions. Notably, CCP110 interacts with two calcium-binding proteins, calmodulin (CaM) and centrin, in vivo . These interactions are critical for cytokinesis regulation. In vitro binding experiments reveal a direct, robust interaction between CP110 and CaM and the existence of multiple high-affinity CaM-binding domains in CP110 .

Native CP110 exists in large complexes (approximately 300 kDa to 3 MDa) that contain both centrin and CaM . CCP110 also interacts with CEP97 to cap the mother centriole and prevent cilia formation . Additionally, the centriolar kinesin Kif24 interacts with CP110 to remodel microtubules and regulate ciliogenesis . These protein-protein interactions are essential for understanding how CCP110 regulates centrosome duplication, ciliogenesis, and cytokinesis.

What are the optimal conditions for using CCP110 antibodies in Western blotting?

For optimal Western blotting conditions when using CCP110 antibodies, follow these methodological guidelines:

• Sample preparation: Use whole cell lysates from cell lines known to express CCP110 (e.g., MOLT4, HeLa, PANC-1, PC-12, RAW264.7) .

• Gel electrophoresis: Run samples on a 5-20% SDS-PAGE gel at 70V (stacking gel) followed by 90V (resolving gel) for 2-3 hours. Load approximately 30 μg of protein per lane under reducing conditions .

• Transfer conditions: Transfer proteins to a nitrocellulose membrane at 150 mA for 50-90 minutes .

• Blocking: Block the membrane with 5% non-fat milk in TBS for 1.5 hours at room temperature .

• Primary antibody incubation: Incubate the membrane with anti-CCP110 antibody at appropriate dilution (typically 0.1-0.5 μg/ml for polyclonal antibodies like A05058-1 or 1:1000 for antibodies like Cell Signaling #12140) overnight at 4°C .

• Washing: Wash the membrane with TBS-0.1% Tween three times, 5 minutes each .

• Secondary antibody: Incubate with appropriate HRP-conjugated secondary antibody (e.g., goat anti-rabbit IgG-HRP) at a dilution of 1:5000 for 1.5 hours at room temperature .

• Detection: Develop using an enhanced chemiluminescent detection system .

The expected molecular weight for CCP110 is approximately 110-113 kDa .

How can I optimize immunofluorescence protocols to detect CCP110 at centrosomes?

To optimize immunofluorescence protocols for detecting CCP110 at centrosomes, follow these methodological steps:

• Cell preparation: Culture cells (e.g., HeLa, U2OS) on glass coverslips to 70-80% confluence.

• Fixation: Fix cells with 4% paraformaldehyde for 10-15 minutes at room temperature. Alternatively, for better preservation of centrosomal structures, use methanol fixation at -20°C for 10 minutes.

• Permeabilization: Permeabilize cells with 0.5% Triton X-100 in PBS for 5-10 minutes at room temperature (skip this step if methanol fixation was used).

• Antigen retrieval: For optimal detection, perform enzyme antigen retrieval using IHC enzyme antigen retrieval reagent for 15 minutes .

• Blocking: Block with 10% goat serum (or serum from the species of the secondary antibody) for 1 hour at room temperature .

• Primary antibody: Incubate with anti-CCP110 antibody at the appropriate concentration (e.g., 5 μg/ml of A05058-1) overnight at 4°C .

• Secondary antibody: Incubate with fluorophore-conjugated secondary antibody (e.g., Cy3-conjugated goat anti-rabbit IgG at 1:500 dilution) for 30 minutes at 37°C .

• Counterstaining: Counterstain nuclei with DAPI.

• Co-staining: To confirm centrosomal localization, co-stain with other centrosomal markers such as γ-tubulin or centrin.

• Visualization: Visualize using a fluorescence microscope with appropriate filter sets .

This protocol can be further optimized based on specific cell types and experimental requirements.

What are the best validation methods to confirm CCP110 antibody specificity?

To rigorously validate CCP110 antibody specificity, employ multiple complementary approaches:

• RNAi-mediated depletion: Perform siRNA knockdown of CCP110 and demonstrate reduction in antibody signal via Western blot and immunofluorescence. This provides strong evidence for antibody specificity .

• Expression of recombinant protein: Overexpress tagged CCP110 (e.g., GFP-CCP110) and confirm co-localization with the antibody signal in immunofluorescence studies and detection of the appropriate sized band in Western blots .

• Multiple antibody validation: Use multiple antibodies targeting different epitopes within CCP110 and confirm consistent localization and detection patterns .

• Immunoprecipitation followed by mass spectrometry: Perform IP with the CCP110 antibody and confirm the presence of CCP110 peptides by mass spectrometry.

• Cross-species reactivity analysis: Test the antibody against CCP110 from different species with known sequence homology to confirm expected cross-reactivity patterns .

• Peptide competition assay: Pre-incubate the antibody with the immunizing peptide and demonstrate loss of signal in Western blot or immunofluorescence.

• Testing in multiple cell lines: Validate antibody performance across various cell types known to express CCP110 (e.g., HeLa, U2OS, MOLT4, PANC-1) .

These rigorous validation approaches ensure confidence in antibody specificity and experimental results.

Why might I observe multiple bands when performing Western blot with CCP110 antibodies?

Multiple bands in CCP110 Western blots may occur for several reasons:

• Post-translational modifications: CCP110 is a substrate for cyclin-dependent kinases (CDKs) and undergoes phosphorylation during the cell cycle, potentially causing mobility shifts . Different phosphorylation states may result in multiple bands ranging from the calculated molecular weight (39 kDa) to the observed 110-113 kDa.

• Protein isoforms: At least two isoforms of CCP110 are known to exist. Some antibodies, like the Nordic Biosite 356-600-401-AG2, detect only the longer isoform . The presence of multiple isoforms may result in multiple bands.

• Proteolytic degradation: Incomplete protease inhibition during sample preparation can lead to degradation products appearing as lower molecular weight bands.

• Cross-reactivity: Despite claims of no cross-reactivity with other proteins , antibodies may sometimes detect proteins with similar epitopes.

• Antibody specificity issues: Some antibodies may have batch-to-batch variation or decreased specificity under certain conditions.

To address these issues:

• Use fresh samples with complete protease inhibitor cocktails

• Optimize sample preparation protocols

• Consider using phosphatase inhibitors if studying phosphorylated forms

• Compare results with multiple antibodies targeting different epitopes

• Include appropriate positive controls (e.g., recombinant CCP110)

• Consider using CCP110 knockout/knockdown samples as negative controls

How can I interpret conflicting localization patterns of CCP110 between different studies?

Conflicting localization patterns of CCP110 across different studies can be explained and addressed through several methodological considerations:

• Cell cycle-dependent localization: CCP110 localization changes throughout the cell cycle. While predominantly centrosomal, its association with calmodulin (CaM) occurs throughout the cell cycle but becomes particularly crucial during cytokinesis . Studies focusing on different cell cycle stages may report varying localization patterns.

• Fixation artifacts: Different fixation methods (paraformaldehyde versus methanol) can significantly affect epitope accessibility and observed localization patterns. Methanol fixation often better preserves centrosomal structures but may disrupt some protein-protein interactions.

• Antibody epitope accessibility: Different antibodies target distinct epitopes that may be differentially accessible depending on CCP110's interaction partners or conformational states. For example, antibodies targeting epitopes involved in CaM binding might show altered staining patterns when that interaction is occurring.

• Technical differences in imaging: Variation in microscopy techniques, from conventional fluorescence to super-resolution approaches, can yield different observations about precise protein localization.

• Cell type-specific differences: CCP110 may exhibit subtly different localization patterns in different cell types based on expression levels of interaction partners.

To resolve conflicting observations:

• Clearly document cell cycle stages in your experiments

• Compare multiple fixation protocols

• Use multiple antibodies targeting different CCP110 regions

• Consider co-localization studies with established centrosomal markers

• Employ super-resolution microscopy techniques for more precise localization

• Validate key findings across multiple cell types

What strategies can improve signal-to-noise ratio when detecting CCP110 in immunohistochemistry?

To improve signal-to-noise ratio when detecting CCP110 in immunohistochemistry (IHC), implement these methodological strategies:

• Optimal antigen retrieval: Heat-mediated antigen retrieval in EDTA buffer (pH 8.0) has been validated for CCP110 detection in paraffin-embedded sections . This enhances epitope accessibility while preserving tissue morphology.

• Appropriate antibody selection: Use antibodies specifically validated for IHC applications, such as the Anti-CP110/CCP110 Picoband (A05058-1) or CCP110 Antibody (356-600-401-AG2) .

• Antibody concentration optimization: Titrate antibody concentrations to determine optimal working dilutions. For example, A05058-1 is recommended at 2-5 μg/ml for IHC applications .

• Blocking optimization: Use 10% serum from the same species as the secondary antibody (e.g., 10% goat serum) to effectively block non-specific binding sites .

• Detection system selection: For CCP110, peroxidase-conjugated secondary antibodies with DAB chromogen have been successfully employed. Consider using amplification systems like HRP-conjugated polymer detection kits for enhanced sensitivity .

• Incubation conditions: Extend primary antibody incubation to overnight at 4°C rather than shorter incubations at higher temperatures .

• Washing optimization: Include additional and longer washing steps with TBS-Tween to reduce background staining.

• Positive and negative controls: Include known positive tissues (e.g., human breast cancer tissue, colorectal adenocarcinoma tissue, testicular germ cell tumor tissue) and antibody omission controls .

• Counterstaining adjustment: Optimize hematoxylin counterstaining time to provide adequate nuclear detail without obscuring specific CCP110 staining.

These strategies, when systematically implemented and optimized, can significantly improve signal-to-noise ratio in CCP110 immunohistochemistry.

How can CCP110 antibodies be used to investigate the relationship between centrosome dysfunction and ciliopathies?

CCP110 antibodies offer powerful tools to investigate the relationship between centrosome dysfunction and ciliopathies through several advanced experimental approaches:

• Spatiotemporal dynamics analysis: Using super-resolution microscopy with CCP110 antibodies, researchers can precisely track CCP110 positioning during the transition from centrosome to basal body in ciliated cells. This approach can reveal dysregulation in ciliopathy models where the removal of CCP110 from the mother centriole—a prerequisite for ciliogenesis—might be impaired .

• Interaction partner studies: CCP110 antibodies can be employed in co-immunoprecipitation experiments to identify altered interaction networks in ciliopathy models. Recent research has shown that CCP110 forms complexes with various proteins including CEP97 and Kif24 to regulate ciliogenesis . Changes in these interactions may underlie certain ciliopathies.

• Post-translational modification mapping: Using CCP110 antibodies in combination with phospho-specific antibodies, researchers can investigate how altered phosphorylation or ubiquitination of CCP110 affects its function. CCP110 is a target of SCF (Cyclin F) ubiquitin ligase complex, suggesting that this complex controls centrosome homeostasis and mitotic fidelity through CCP110 degradation .

• In vivo ciliopathy models: CCP110 antibodies can be used in immunohistochemistry of tissues from ciliopathy models (e.g., mouse models of Joubert syndrome or Bardet-Biedl syndrome) to determine if CCP110 removal from mother centrioles is affected in specific cell types or developmental stages.

• Rescue experiments analysis: In cells from ciliopathy patients, researchers can perform rescue experiments with wild-type or mutant proteins involved in CCP110 regulation and use CCP110 antibodies to assess normalization of centrosome-to-basal body conversion.

• Co-localization with ciliopathy proteins: CCP110 antibodies can be used in co-localization studies with proteins encoded by ciliopathy genes (e.g., WDR44, which causes a spectrum of ciliopathy by impairing ciliogenesis initiation ) to identify mechanistic connections.

This multifaceted approach can provide critical insights into how dysregulation of CCP110 contributes to ciliopathies and may identify novel therapeutic targets.

What methodological approaches can be used to study CCP110's role in the calcium signaling pathway during centrosome duplication and cytokinesis?

To investigate CCP110's role in calcium signaling during centrosome duplication and cytokinesis, implement these advanced methodological approaches:

• FRET-based calcium sensors with CCP110 co-localization: Deploy genetically encoded calcium indicators (GECIs) targeted to centrosomes alongside fluorescently tagged CCP110 to simultaneously monitor local calcium fluctuations and CCP110 dynamics during centrosome duplication and cytokinesis.

• CCP110 calcium-binding mutant analysis: Generate CCP110 mutants defective in calcium binding based on identified calmodulin (CaM) binding domains. Experimental evidence shows that CP110 has multiple high-affinity CaM-binding domains, and expression of a CP110 mutant unable to bind CaM promotes cytokinesis failure and binucleate cell formation . Comparing phenotypes between wild-type and mutant CCP110 can elucidate calcium dependency.

• Calcium chelation experiments: Use cell-permeable calcium chelators like BAPTA-AM at doses that don't completely block cell division, then assess changes in CCP110 localization, phosphorylation state, and interaction partners using antibody-based approaches.

• Proximity ligation assays (PLA): Employ PLA using antibodies against CCP110 and calcium-binding proteins (CaM and centrin) to visualize and quantify their interactions under different calcium conditions and cell cycle stages .

• Calcium ionophore treatments: Treat cells with calcium ionophores to artificially elevate intracellular calcium levels and assess CCP110 modifications and centrosomal localization using specific antibodies.

• Live-cell imaging with calcium modulation: Combine live-cell imaging of fluorescently tagged CCP110 with controlled calcium modulation to directly observe how calcium fluctuations affect CCP110 dynamics during centrosome duplication and cytokinesis.

• Mass spectrometry of CCP110 complexes: Immunoprecipitate CCP110 under different calcium conditions and perform mass spectrometry to identify calcium-dependent changes in the composition of CCP110 complexes, which normally exist in large (∼300 kDa to 3 MDa) assemblies containing both centrin and CaM .

These methodological approaches can systematically dissect the role of CCP110 in calcium-dependent processes at the centrosome and during cytokinesis.

How can novel CP110 antibodies be developed to study tissue-specific or developmental stage-specific functions of this protein?

Development of novel CP110 antibodies for tissue-specific or developmental stage-specific research requires sophisticated methodological approaches:

• Isoform-specific antibody development: Design antibodies targeting unique regions of CCP110 isoforms that may be differentially expressed across tissues or developmental stages. At least two isoforms are known to exist, with some current antibodies detecting only the longer isoform . Developing isoform-specific antibodies would enable precise tracking of expression patterns.

• Post-translational modification (PTM)-specific antibodies: Generate antibodies that specifically recognize phosphorylated, ubiquitinated, or otherwise modified forms of CCP110. Since CCP110 is a substrate for cyclin-dependent kinases (CDKs) and the SCF (Cyclin F) ubiquitin ligase complex , PTM-specific antibodies could reveal tissue-specific or developmental regulation.

• Conformation-specific antibodies: Develop antibodies that recognize specific conformational states of CCP110, particularly those associated with its various binding partners. This approach could distinguish between CCP110 in complex with CEP97 (inhibiting ciliogenesis) versus CCP110 in other functional states.

• Recombinant antibody engineering: Utilize phage display or yeast display technologies to engineer recombinant antibody fragments (Fabs or scFvs) with enhanced specificity for CCP110 epitopes that may be masked in certain tissues or developmental contexts.

• Conditional fluorescent tagging: Develop tissue-specific or developmental stage-specific Cre-recombinase mouse models with floxed fluorescent protein tags inserted into the endogenous CCP110 locus. While not antibodies per se, these genetic tools would complement antibody-based approaches by allowing visualization of endogenous CCP110 expression patterns.

• Nanobody development: Generate camelid-derived single-domain antibodies (nanobodies) against CCP110, which due to their small size may access epitopes unreachable by conventional antibodies, potentially revealing previously unrecognized pools of CCP110.

• Cross-species epitope analysis: Identify evolutionarily conserved and divergent regions of CCP110 across species and develop antibodies targeting these specific regions to facilitate comparative studies of CCP110 function across model organisms.

These novel antibody development strategies would significantly advance our understanding of tissue-specific and developmental functions of CCP110 in normal development and disease states.

What are the most promising experimental designs to investigate the potential role of CCP110 dysregulation in cancer progression?

To investigate CCP110 dysregulation in cancer progression, implement these sophisticated experimental designs:

• Multi-cancer tissue microarray analysis: Use validated CCP110 antibodies on tissue microarrays spanning multiple cancer types and their corresponding normal tissues. The documented positive tissues for CCP110 antibodies include human breast cancer tissue, colorectal adenocarcinoma tissue, and testicular germ cell tumor tissue . Quantify expression levels and correlate with clinicopathological features to identify cancer types where CCP110 dysregulation is particularly relevant.

• CRISPR/Cas9-mediated CCP110 modulation in cancer models: Generate isogenic cancer cell lines with CCP110 knockout, knockdown, or overexpression using CRISPR/Cas9 technology. Assess effects on centrosome numbers, mitotic fidelity, genomic stability, and hallmarks of cancer (proliferation, invasion, anchorage-independent growth) using antibody-based detection methods.

• Patient-derived xenograft (PDX) studies: Establish PDX models from tumors with varying CCP110 expression levels (as determined by antibody staining). Monitor tumor growth, metastatic potential, and response to therapies, particularly those targeting cell cycle checkpoints or centrosome amplification.

• Live-cell imaging of CCP110 dynamics in cancer cells: Using cell lines stably expressing fluorescently tagged CCP110, perform live-cell imaging during cell division in normal versus cancer cells. Compare centrosomal localization patterns, removal kinetics during ciliogenesis, and protein stability using complementary antibody-based approaches for validation.

• Interaction proteomics in cancer contexts: Perform immunoprecipitation with CCP110 antibodies followed by mass spectrometry in normal versus cancer cells to identify cancer-specific alterations in CCP110 interaction networks, particularly focusing on interactions with oncogenes or tumor suppressors.

• Centrosome amplification correlation studies: In cancer samples with centrosome amplification, use CCP110 antibodies alongside markers of centrosome amplification (e.g., γ-tubulin, centrin) to determine if CCP110 levels correlate with the degree of amplification and chromosomal instability.

• Therapeutic targeting evaluation: Test compounds that indirectly modulate CCP110 levels or activity (e.g., CDK inhibitors, which may affect CCP110 phosphorylation) in CCP110-high versus CCP110-low cancer models. Use CCP110 antibodies to monitor effects on protein levels, localization, and modification states.

• Correlation with cancer stem cell markers: In tumors with cancer stem cell populations, investigate whether CCP110 levels (detected by antibody staining) correlate with stemness markers, potentially indicating a role in maintaining cancer stem cell properties.

These experimental approaches would provide comprehensive insights into how CCP110 dysregulation contributes to cancer progression and potential therapeutic vulnerabilities.