CEP15 Antibody

What is CEP15 and how does it differ from CEP152?

• CEP15 (Centrosomal protein 15) - A centrosomal protein involved in cell division

• CEP152 (Centrosomal protein 152kDa) - A different centrosomal protein necessary for centrosome duplication

• CEP15 (Chromosomal enumeration probe) - A probe targeting the centromeric region of chromosome 15

This distinction is crucial as researchers must ensure they're working with antibodies specific to their target of interest. CEP152 is more extensively characterized in the literature and has multiple commercially available antibodies , while specific antibodies against human CEP15 protein are less common.

What are the molecular characteristics of CEP15 compared to CEP152?

CEP15 functions as a component of the centrosome involved in cell cycle regulation and spatial organization of the cell . Meanwhile, CEP152 (189 kDa calculated molecular weight) serves as a molecular scaffold that facilitates the interaction of PLK4 and CENPJ, two molecules involved in centriole formation . CEP152 is necessary for centrosome duplication, with its function involving CEP63, CDK5RAP2, and WDR62 through a stepwise assembled complex at the centrosome that recruits CDK2 required for centriole duplication . CEP152 also plays a key role in deuterosome-mediated centriole amplification in multiciliated cells that can generate more than 100 centrioles .

Applications and Methodological Considerations

For immunofluorescence applications with CEP152 antibodies, the following protocol has been validated:

• Sample preparation: Fix cells in 2% paraformaldehyde/culture medium at 37°C for 30 min

• Antibody dilution: Use CEP152 antibody (ab183911) at 1/2000 dilution

• Costaining: For optimal visualization of cellular structures, costain with:

  • Hoechst 33342 (blue) for nuclear visualization

  • Alpha 1b tubulin antibody at 1/5000 dilution (red) for microtubule visualization

For experiments requiring subcellular localization studies, centrosomal markers should be used to confirm the expected localization pattern of CEP15/CEP152.

How should CEP15/CEP152 antibodies be validated to ensure specificity?

Antibody validation is crucial to ensure experimental reproducibility. According to current standards, CEP15/CEP152 antibodies should be validated using multiple approaches :

• Orthogonal methods: Compare antibody-based measurements with measurements based on an antibody-independent method

• Genetic knockdown: Test antibody performance in samples with reduced target expression

• Recombinant expression: Validate antibody using samples with overexpressed target

• Independent antibodies: Use multiple antibodies targeting different epitopes of the same protein

• Capture mass spectrometry: Identify proteins recognized by the antibody

For CEP152 specifically, researchers should be aware of potential cross-reactivity issues. For example, a mouse monoclonal antibody A17 for cyclin-dependent kinase 1 (CDK1) has been shown to cross-react with CEP152 protein in both Western blotting and immunocytochemistry applications . This highlights the importance of rigorous validation.

What controls should be included when working with CEP15/CEP152 antibodies?

To ensure experimental validity when working with CEP15/CEP152 antibodies, researchers should include:

• Positive controls: Cell lines known to express the target protein (e.g., HEK-293T, HeLa for CEP152)

• Negative controls:

  • Primary antibody omission

  • Use of isotype control antibodies

  • When possible, samples from knockout/knockdown models

• Peptide competition assays: To demonstrate binding specificity, as shown in ab110825 validation where CEP152 staining was eliminated by pre-incubation with the immunizing peptide

• Cross-reactivity controls: Especially important given documented cases of cross-reactivity between antibodies for cell cycle proteins and CEP152

How do you address cross-reactivity issues when using CEP152 antibodies?

Cross-reactivity is a significant concern with CEP152 antibodies. The following approaches can help address this issue:

• Molecular weight verification: CEP152 has isoforms with molecular weights of approximately 189 kDa, 152 kDa, 110 kDa, and 66 kDa . Verify that your antibody detects bands at the expected molecular weight.

• Epitope mapping: Choose antibodies targeting unique regions of CEP152:

  • N-terminal antibodies (e.g., ABE1856)

  • C-terminal antibodies

  • Antibodies against internal regions (e.g., aa 50-300 for ab183911)

• Validation in knockout/knockdown systems: Test antibody specificity in systems where CEP152 expression has been reduced or eliminated .

• Pre-adsorption controls: Pre-incubate the antibody with its immunizing peptide to confirm binding specificity .

• Multiple antibody approach: Use multiple antibodies targeting different epitopes to confirm results .

What are common technical issues when using CEP15/CEP152 antibodies in Western blotting?

When performing Western blotting with CEP15/CEP152 antibodies, researchers may encounter several technical challenges:

• Multiple bands: CEP152 has multiple isoforms (189 kDa, 152 kDa, 110 kDa, and 66 kDa) . The observed molecular weight of 66 kDa reported for some CEP152 antibodies may represent a specific isoform or degradation product.

• Sample preparation: For optimal results with CEP152 antibodies:

  • Use whole cell lysates from appropriate cell lines (HEK-293T, HeLa)

  • Load adequate protein (30 µg per lane recommended)

  • Use 5% SDS-PAGE gels to properly resolve high molecular weight proteins

• Antibody dilution: Optimal dilutions vary by antibody:

  • ab183911: 1/1000 for Western blot

  • 21815-1-AP: 1/200-1/1000 for Western blot

• Blocking and washing conditions: Follow manufacturer's recommendations for each specific antibody to minimize background and enhance specific signal.

How can CEP15/CEP152 antibodies be used in studying centrosome biology and cell cycle regulation?

CEP152 plays critical roles in centrosome biology that can be studied using specific antibodies:

• Centrosome duplication studies: CEP152 is necessary for centrosome duplication through interactions with CEP63, CDK5RAP2, and WDR62 . Antibodies can be used to:

  • Track protein localization during cell cycle progression

  • Immunoprecipitate protein complexes to identify interaction partners

  • Analyze post-translational modifications that regulate function

• PLK4-mediated centriole formation: CEP152 acts as a molecular scaffold facilitating the interaction of PLK4 and CENPJ . Researchers can use antibodies to:

  • Study colocalization with PLK4 and CENPJ

  • Analyze temporal dynamics of complex formation

  • Investigate how CEP152 repositions PLK4 at the outer boundary of forming CEP152 ring structures

• Centriole amplification mechanisms: CEP152 plays a key role in deuterosome-mediated centriole amplification in multiciliated cells . Antibodies can help:

  • Visualize centriole amplification processes

  • Quantify CEP152 levels in correlation with centriole number

  • Study overexpression effects that can drive amplification of centrioles

What are the considerations for using CEP15/CEP152 antibodies in studying genetic disorders?

CEP152 has been implicated in genetic disorders, making antibodies valuable tools for disease research:

• Seckel syndrome and microcephaly: CEP152 is associated with primary autosomal recessive microcephaly (MCPH4, MCPH9) and Seckel syndrome (SCKL5) . Researchers should:

  • Select antibodies validated for detecting disease-associated mutations

  • Consider using patient-derived samples to study pathological mechanisms

  • Include appropriate controls from unaffected individuals

• Cancer research applications: Centrosome abnormalities are common in cancer, and CEP152 overexpression can drive amplification of centrioles . When studying cancer samples:

  • Consider tissue-specific expression patterns

  • Use antibodies validated for the specific sample type (e.g., tissue sections, cell lines)

  • Correlate CEP152 expression with clinical parameters

• Genomic integrity studies: CEP152 functions as a regulator of genomic integrity and cellular response to DNA damage through ATR-mediated checkpoint signaling . Researchers can:

  • Study CEP152 localization in response to DNA damage

  • Analyze interactions with ATR and CENPJ in cellular responses to DNA damage

  • Investigate connections between centrosome abnormalities and genomic instability

How are CEP15/CEP152 antibodies being used in new research approaches?

Recent advances in microscopy and proteomics are expanding the applications of CEP15/CEP152 antibodies:

• Super-resolution microscopy: Techniques like STORM and STED microscopy combined with highly specific antibodies can reveal detailed subcellular localization and protein interactions at the centrosome.

• Proximity labeling approaches: Antibodies can be used in conjunction with techniques like BioID or APEX to identify proteins in close proximity to CEP15/CEP152 in living cells.

• Single-cell analysis: CEP15/CEP152 antibodies are being adapted for single-cell proteomics approaches to understand cell-to-cell variation in centrosome composition and function.

• Electron microscopy applications: CEP152 antibodies have been validated for electron microscopy (EM) , enabling ultrastructural studies of centrosome organization.

What quality control metrics should researchers apply when selecting CEP15/CEP152 antibodies?

When selecting antibodies for CEP15/CEP152 research, consider these quality control metrics:

• Application-specific validation: Ensure the antibody has been validated for your specific application (WB, IF, IHC, IP) .

• Lot-to-lot consistency: Request lot-specific validation data from manufacturers to ensure consistency.

• Citation record: Review publications using the same antibody for similar applications.

• Reproducibility data: Look for antibodies validated using multiple approaches as outlined by the International Working Group for Antibody Validation .

• Epitope information: Select antibodies with well-defined epitopes that are:

  • Accessible in your experimental conditions

  • Conserved across species if performing cross-species studies

  • Not subject to post-translational modifications that might affect antibody binding

• Storage and handling: Follow manufacturer recommendations for storage (typically -20°C) and avoid repeated freeze-thaw cycles to maintain antibody performance.