CEP152 Antibody

What is CEP152 and why is it important in cellular research?

CEP152 (centrosomal protein 152kDa), also known as KIAA0912, is a critical component of the centrosome with multiple vital cellular functions. It serves as a regulator of genomic integrity and mediates cellular responses to DNA damage through ATR-mediated checkpoint signaling. CEP152 is essential for centrosome duplication, functioning as a molecular scaffold that facilitates the interaction between PLK4 and CENPJ, two key molecules involved in centriole formation . Research has shown that CEP152, along with ATR and CENPJ, plays a crucial role in controlling cellular responses to DNA damage . The protein exhibits multiple isoforms with molecular weights of 189 kDa, 152 kDa, 110 kDa, and 66 kDa, with the 66 kDa variant commonly observed in experimental settings .

What applications are CEP152 antibodies suitable for?

Based on validated research protocols, CEP152 antibodies have been successfully applied in multiple experimental techniques:

Researchers should note that optimal dilutions may vary by sample type and specific antibody, and titration is recommended for each experimental system to obtain optimal results .

What species reactivity can be expected with commercially available CEP152 antibodies?

Different CEP152 antibodies show varying species reactivity profiles:

When selecting an antibody for your research, consider both tested and predicted reactivity, especially for cross-species studies. While some antibodies have been validated in human and mouse samples, others may have broader predicted reactivity based on sequence homology .

What are the recommended protocols for CEP152 detection by immunofluorescence?

For optimal immunofluorescence detection of CEP152 at the centrosome:

• Sample Preparation: Fix cells in 2% paraformaldehyde in culture medium at 37°C for 30 minutes . For alternative fixation methods, cold methanol fixation has also been successful for centrosomal proteins.

• Antibody Dilution:

  • For Abcam ab183911: Use at 1/2000 dilution

  • For other antibodies: Refer to manufacturer's recommended dilutions, typically starting at 1:100-1:500

• Co-staining Recommendations: Co-stain with centrosomal markers or centriolar markers for proper localization:

  • α-tubulin (1/5000) to visualize microtubules

  • Hoechst 33342 for nuclear staining

  • Centrin for distal end centriole marking

  • γ-tubulin for pericentriolar material

• Imaging Considerations: For optimal resolution of centrosomal structures, deconvolution microscopy is recommended to distinguish CEP152 from other centrosomal proteins like centrin, γ-tubulin, and Sas6 .

Research has demonstrated that CEP152 localizes to the centrosome throughout the cell cycle, with distribution distinct from both centrin and γ-tubulin in G1 phase centrosomes .

How should I optimize Western blot protocols for CEP152 detection?

For successful Western blot detection of CEP152:

• Sample Preparation:

  • Whole cell lysates from human cell lines (HEK-293T, HeLa) have been successfully used

  • Mouse brain tissue is recommended as a positive control

  • Load approximately 30 μg protein per lane

• Gel Selection and Transfer:

  • Use 5% SDS-PAGE gels to properly resolve the high molecular weight CEP152 protein

  • Standard transfer protocols for high molecular weight proteins are recommended

• Antibody Dilution and Detection:

  • Primary antibody: 1:200-1:1000 (varies by manufacturer)

  • Secondary antibody: Anti-Rabbit IgG (most CEP152 antibodies are rabbit polyclonal)

• Expected Banding Pattern:

  • Calculated molecular weight is 189 kDa

  • Commonly observed band at 66 kDa in experimental settings

  • Be aware of possible additional bands representing other isoforms (152 kDa, 110 kDa)

For optimal results, perform antibody titration experiments specific to your sample type and protein extraction method.

What controls should be included when working with CEP152 antibodies?

Comprehensive experimental design requires appropriate controls:

• Positive Controls:

  • Western blot: Mouse brain tissue is a validated positive control

  • IHC: Human brain tissue is recommended

  • IF/ICC: HeLa cells show reliable CEP152 centrosomal staining

• Negative Controls:

  • Primary antibody omission

  • CEP152 knockdown/knockout samples for antibody specificity validation

  • Non-expressing cell types or tissues

• Validation Controls:

  • Peptide competition assays

  • Use of multiple antibodies recognizing different epitopes

  • Parallel detection with tagged recombinant protein expression

• Localization Controls for IF:

  • Co-staining with established centrosomal markers:

    • γ-tubulin (pericentriolar material)

    • Centrin (distal end of centriole)

    • Sas6 (for duplicated centriole pairs)

How can CEP152 antibodies be used to study centrosome duplication and centriole formation?

CEP152 plays a critical role in centrosome duplication, making its antibodies valuable tools for studying this process:

• Tracking Centrosome Duplication Cycles:

  • Use immunofluorescence with CEP152 antibodies to monitor centrosome numbers through the cell cycle

  • Co-stain with centrin to count centrioles and assess duplication status

  • Track CEP152 recruitment during different phases of the duplication cycle

• Functional Studies:

  • Combine with PLK4 and CPAP antibodies to study their sequential recruitment to centrosomes

  • Use in conjunction with cell cycle markers to correlate CEP152 dynamics with specific cell cycle phases

  • Map interaction domains through co-immunoprecipitation experiments with truncated protein variants

• Experimental Approaches:

  • Synchronize cells and analyze CEP152 localization at specific cell cycle stages

  • Monitor CEP152 after PLK4 overexpression to study centriole overduplication

  • Examine effects of DNA damage on CEP152 localization and function

Research has demonstrated that CEP152 depletion leads to a stepwise decrease in centriole number over time, consistent with defects in centriole duplication followed by segregation errors in mitosis . Studies have also shown that CEP152 acts as a scaffold, with distinct regions mediating interactions with PLK4 (N-terminal) and CPAP, while centrosomal localization is determined by its C-terminal region .

What is the role of CEP152 in DNA damage response, and how can antibodies help investigate this function?

CEP152 serves as a critical genome maintenance protein with connections to DNA damage response pathways:

• Experimental Approaches:

  • Use CEP152 antibodies in combination with DNA damage markers (γH2AX) after inducing replicative stress with hydroxyurea

  • Examine co-localization with ATR-pathway components

  • Monitor CEP152 dynamics after various genotoxic treatments

• Functional Analyses:

  • Combine CEP152 antibodies with cell cycle analysis to detect S-phase entry delays

  • Perform sister chromatid exchange frequency measurements after CEP152 depletion

  • Investigate checkpoint activation markers in CEP152-deficient cells

• Research Findings:

  • CEP152 knockdown delays S-phase entry

  • Fewer CEP152-Seckel cells progress to G2/M phase

  • CEP152 deficiency leads to increased chromosome instability

  • Hydroxyurea treatment of CEP152-deficient cells shows substantially increased γH2AX levels

Research has revealed that CEP152 interacts with CINP (CDK2-interacting protein), creating a potential link between centrosome function and DNA replication/repair processes. Combined with evidence that CEP152 deficiency increases replicative stress and activates ATM-dependent DNA damage responses, these findings suggest multiple mechanisms by which CEP152 maintains genomic integrity .

How can CEP152 antibodies be used to investigate protein-protein interactions at the centrosome?

CEP152 functions as a scaffold protein at the centrosome, making it ideal for studying centrosomal protein networks:

• Co-immunoprecipitation Studies:

  • Use CEP152 antibodies to pull down interacting partners

  • Perform reverse co-IPs with antibodies against putative interactors

  • Validate endogenous protein interactions in centrosome-enriched fractions

• Domain Mapping Experiments:

  • Based on published research, CEP152 interacts with:

    • PLK4 via its N-terminal domain (residues 1-512)

    • CPAP via residues 513-1074

    • Self-interaction through its SMC-like domains

• Localization Studies:

  • Use structured illumination or deconvolution microscopy with CEP152 antibodies to resolve:

    • Relative positioning of CEP152 and interacting partners at the centrosome

    • Temporal recruitment patterns during centrosome duplication

    • Effects of mutant protein expression on partner localization

• Functional Validation:

  • Express dominant-negative fragments (e.g., CEP152 1-512) to disrupt specific interactions

  • Analyze effects on partner protein recruitment to centrosomes

  • Assess consequences for centriole duplication and cell division

Research has revealed that CEP152 functions as a scaffold for both PLK4 and CPAP, with distinct domains mediating these interactions. Notably, while the N-terminal region binds PLK4, centrosomal localization is determined by the C-terminal domain, providing insight into how CEP152 serves as an assembly platform at the centrosome .

Why might I observe different molecular weights for CEP152 in Western blot experiments?

The discrepancy between calculated and observed molecular weights of CEP152 is a common issue with several explanations:

• Multiple Isoforms:

  • CEP152 has several isoforms with different molecular weights:

    • Calculated full-length: 189 kDa

    • Commonly observed: 66 kDa

    • Additional reported isoforms: 152 kDa, 110 kDa

• Antibody Epitope Specificity:

  • Different antibodies target distinct regions and may preferentially detect specific isoforms

  • Check the immunogen information provided by manufacturers:

    • Proteintech 21815-1-AP: Peptide immunogen

    • Abcam ab183911: Recombinant fragment (aa 50-300)

• Technical Considerations:

  • Large proteins may experience incomplete transfer during Western blotting

  • Higher percentage gels may not properly resolve high molecular weight proteins

  • Post-translational modifications can alter migration patterns

• Recommended Approach:

  • Use 5% SDS-PAGE gels for better resolution of high molecular weight proteins

  • Include multiple positive controls

  • When possible, validate with CEP152 knockdown/knockout samples

  • Consider using multiple antibodies that target different epitopes

These variations highlight the importance of proper experimental controls and awareness of isoform diversity when interpreting CEP152 Western blot results.

How can I differentiate between specific and non-specific staining in CEP152 immunofluorescence experiments?

Ensuring specificity in CEP152 immunofluorescence studies requires careful controls and analysis:

• Characteristic CEP152 Localization Pattern:

  • Authentic CEP152 staining should show:

    • Distinct centrosomal localization throughout the cell cycle

    • Distribution pattern distinct from centrin (distal end marker)

    • Distribution pattern distinct from γ-tubulin (PCM marker)

• Validation Controls:

  • Perform siRNA/shRNA-mediated knockdown of CEP152 to confirm signal reduction

  • Compare multiple antibodies targeting different epitopes

  • Include peptide competition controls if available

• Co-localization Experiments:

  • Co-stain with established centrosomal markers:

    • Centrin (distal centriole end)

    • γ-tubulin (pericentriolar material)

    • Sas6 (for duplicated centriole pairs)

  • Use high-resolution or deconvolution microscopy to properly resolve centrosomal structures

• Technical Considerations:

  • Optimize fixation methods (2% paraformaldehyde/culture medium at 37°C for 30 min has been validated)

  • Test multiple antibody dilutions

  • Consider antigen retrieval methods for tissue sections (TE buffer pH 9.0 recommended for human brain tissue)

When interpreting results, remember that CEP152 exhibits only partial overlap with Sas6 in duplicated centriole pairs, and its distribution relative to other centrosomal proteins provides important spatial information about centrosome organization .

What methodological considerations should be addressed when using CEP152 antibodies in centrosome duplication studies?

When investigating centrosome duplication with CEP152 antibodies, several methodological aspects require careful attention:

• Cell Synchronization Strategies:

  • S-phase arrest with hydroxyurea or thymidine can be used to study centrosome reduplication

  • Mitotic shake-off followed by release enables examination of synchronized duplication

  • Consider that CEP152 depletion affects cell cycle progression itself

• Quantification Methods:

  • Count centriole numbers in mitotic cells using centrin as a marker

  • Distinguish between paired and single centrioles

  • Use z-stack imaging to ensure all centrioles are captured

  • Analyze sufficient cell numbers (typically >100 cells per condition)

• Temporal Considerations:

  • CEP152 depletion experiments should extend to 72-96 hours to observe maximum effects

  • Stepwise decrease in centriole numbers indicates duplication defects

  • For mitotic phenotypes, analyze 72 hours post-depletion when defects become prominent (~20-25% monopolar spindles)

• Experimental Design:

  • Include appropriate controls:

    • Control siRNA/shRNA treatments

    • Cdk2 siRNA as a positive control for duplication defects

    • Mixed cell populations to ensure internal controls

  • Consider complementation experiments with siRNA-resistant constructs

  • Be aware that expression of N-terminal CEP152 fragments (1-512) can exhibit dominant-negative effects

Research has shown that CEP152 depletion leads to a specific pattern of centriole loss over time, with an increase in monopolar spindles in mitosis and a failure in centriole duplication that can be differentiated from segregation defects by careful quantitative analysis .

How is CEP152 implicated in human disease conditions, and how can antibodies contribute to this research?

CEP152 has important connections to human diseases that can be investigated using antibody-based approaches:

• Disease Associations:

  • Seckel syndrome: A form of primordial dwarfism with microcephaly and DNA damage response defects

  • Primary microcephaly (MCPH): Associated with mutations disrupting centrosome function

  • Giant axonal neuropathy: Linked to cytoskeletal abnormalities

• Research Approaches:

  • Analyze patient-derived cells with CEP152 antibodies to assess:

    • Centrosome abnormalities

    • DNA damage response defects

    • Cell cycle perturbations

  • Compare CEP152 localization and interactions in patient vs. control cells

  • Correlate patient-specific mutations with functional domains of CEP152

• Mechanistic Insights:

  • CEP152 deficiency in Seckel syndrome shows:

    • Delayed S-phase entry

    • Altered ATR-mediated checkpoint activity

    • Increased replicative stress

    • Substantial increase in chromosome instability

    • Elevated γH2AX levels after hydroxyurea treatment

• Technical Considerations:

  • When studying patient material, validate antibody performance in relevant tissues

  • Consider using patient-specific mutations to create cellular models

  • Combine with functional assays such as sister chromatid exchange frequency measurements

Understanding CEP152's dual roles in centrosome function and DNA damage response provides important insights into the molecular basis of microcephaly and growth disorders, with potential implications for broader genomic instability conditions.

What are the latest methodological advances in studying CEP152 protein-protein interactions at the centrosome?

Recent advances have expanded the toolkit for investigating CEP152's role in centrosomal protein networks:

• Advanced Imaging Techniques:

  • Super-resolution microscopy (SIM, STORM) to resolve the precise spatial organization of CEP152 within the centrosome

  • Live-cell imaging with fluorescently tagged proteins to track dynamic interactions

  • FRET/FLIM approaches to detect direct interactions in living cells

• Biochemical Interaction Mapping:

  • BioID or TurboID proximity labeling to identify the CEP152 interactome

  • Combination of yeast two-hybrid screens with complementary immunoprecipitation approaches

  • Validation of constitutive binding to interaction partners like CINP

  • Domain mapping using deletion constructs to identify specific interaction regions:

    • N-terminal region (residues 1-512) for PLK4 binding

    • Residues 513-1074 for CPAP interaction

    • C-terminal region for centrosomal localization

• Functional Validation Methods:

  • Expression of dominant-negative fragments (e.g., CEP152 1-512) to disrupt specific interactions

  • Rescue experiments with structure-guided mutants that disrupt specific interactions

  • Sequential protein recruitment analysis during centrosome duplication

• Stepwise Complex Assembly Model:

  • Research has revealed that CEP152 is part of a stepwise assembled complex at the centrosome

  • This complex includes CEP63, CDK5RAP2, and WDR62

  • The complex recruits CDK2, which is required for centriole duplication

  • CEP152 is proposed to reposition PLK4 at the outer boundary of a newly forming ring structure

These methodological advances provide unprecedented resolution for understanding how CEP152 functions as a centrosomal scaffold and how its interactions with PLK4 and CPAP coordinate centriole duplication.

How can CEP152 antibodies contribute to understanding multiciliated cell development and pathologies?

CEP152 plays a specialized role in multiciliated cells that can be investigated using antibody-based approaches:

• Unique Role in Multiciliated Cells:

  • CEP152 is key in deuterosome-mediated centriole amplification in multiciliated cells

  • These specialized cells can generate more than 100 centrioles

  • Overexpression of CEP152 can drive amplification of centrioles

• Research Applications:

  • Use CEP152 antibodies to study:

    • Deuterosome formation during multiciliogenesis

    • Centriole amplification in multiciliated epithelial cells

    • Potential pathologies affecting multiciliated tissues (respiratory epithelium, ependymal cells)

  • Compare CEP152 localization patterns between standard mitotic centriole duplication and massive centriole amplification

• Experimental Approaches:

  • Immunofluorescence analysis of multiciliated tissues or in vitro differentiated multiciliated cells

  • Co-localization with deuterosome markers and other centriole components

  • Temporal analysis of CEP152 recruitment during stepwise centriole amplification

  • Manipulation of CEP152 levels to assess effects on centriole number and ciliary function

• Pathological Relevance:

  • Dysfunction of multiciliated cells contributes to:

    • Respiratory conditions affecting mucociliary clearance

    • Hydrocephalus (through ependymal cell dysfunction)

    • Infertility (through effects on ciliated reproductive tissues)

  • CEP152 studies may provide insights into the molecular basis of these conditions

This specialized application of CEP152 research highlights the broader importance of this protein beyond its canonical roles in centrosome duplication and genomic integrity.