CEP164 Antibody

What is CEP164 and why is it important in cellular research?

CEP164 is a 164 kDa centrosomal protein comprising 1,460 amino acid residues that localizes specifically to the distal appendages of the mother centriole. It plays a critical role in primary cilium formation, as demonstrated by siRNA experiments showing that only 3.6% of Cep164-depleted cells formed primary cilia compared to 95% of control cells . CEP164 functions in microtubule organization and maintenance for primary cilia formation and is involved in G2/M checkpoint regulation and nuclear divisions . Its expression is typically limited to the mother centriole, making it a valuable marker for distinguishing between mother and daughter centrioles in research applications .

CEP164 is particularly important in research because:

• It serves as a specific marker for mature centrioles

• It plays essential roles in ciliogenesis

• Dysfunction has been implicated in ciliopathies including Bardet-Biedl syndrome and Meckel-Gruber syndrome

• It forms distinctive bar- or ring-like structures at the base of primary cilia that can be visualized through immunofluorescence

What types of CEP164 antibodies are most effective for different applications?

Different CEP164 antibodies target distinct regions of the protein and demonstrate varying efficacy across applications:

For immunofluorescence applications, antibodies targeting the N-terminal fragment (1-298) have been successfully used to identify CEP164's localization to the mother centriole . For Western blot applications, these antibodies typically detect a band of approximately 200 kDa (despite the predicted 164 kDa size), possibly due to the protein's relatively acidic isoelectric point (5.32) .

When selecting an antibody, consider:

• The specific application (WB, IF/ICC, IHC, IP)

• Required species reactivity

• Whether a conjugated antibody would simplify your protocol

• The region of CEP164 that should be targeted based on your research question

How should Western blot protocols be optimized for CEP164 detection?

Optimizing Western blot protocols for CEP164 requires attention to several key factors:

• Sample preparation: For centrosomal proteins like CEP164, enrichment techniques may improve detection. Consider using centrosome purification protocols or specific lysis buffers that preserve large protein complexes .

• Gel selection: Due to CEP164's high molecular weight (~200 kDa observed size), use low percentage (6-8%) polyacrylamide gels or gradient gels to ensure proper resolution .

• Transfer conditions: For large proteins like CEP164:

  • Extend transfer time (overnight at low voltage)

  • Use PVDF membranes rather than nitrocellulose

  • Add SDS (0.1%) to transfer buffer to improve large protein migration

• Antibody dilution: Most CEP164 antibodies work optimally at dilutions between 1:200-1:1000 for Western blot applications . Initial optimization should test multiple dilutions.

• Detection system: Enhanced chemiluminescence systems with extended exposure times may be necessary for optimal detection.

• Controls: Include positive controls such as HEK-293 cell lysate, which has been validated for CEP164 detection .

• Expected results: Anticipate a band at approximately 200 kDa, which is higher than the calculated 164 kDa, due to the protein's acidic properties .

If non-specific bands appear, further optimization of blocking conditions (5% BSA instead of milk) and more stringent washing steps may improve results.

How can CEP164 antibodies be used to distinguish between mother and daughter centrioles?

CEP164 represents an excellent marker for distinguishing mother from daughter centrioles because it specifically localizes to the distal appendages of mature mother centrioles. When implementing this approach:

• Immunofluorescence protocol optimization:

  • Use recommended dilutions of 1:200-1:800 for IF applications

  • Perform co-staining with centrosome markers such as γ-tubulin to identify the centrosomal region first

  • For optimal visualization, super-resolution microscopy techniques may provide better resolution of the distinct ring-like structure

• Expected staining pattern:

  • CEP164 appears as a distinctive bar- or ring-like structure (depending on viewing angle) at the mature centriole

  • In a typical cell with two centrioles, only one will show CEP164 staining

  • In cells with primary cilia, CEP164 localizes to the single centriole at the base of the cilium

• Experimental verification:

  • In studies of primary cilia formation, researchers observed that CEP164 "always associated with the one centriole that was located at the base of the PC, in agreement with the fact that only the mature centriole is able to initiate PC formation"

  • The ring-like structure represents the distal appendages of the mother centriole, which are absent in daughter centrioles

• Additional markers for confirmation:

  • Consider co-staining with other mother centriole-specific proteins

  • Include acetylated tubulin staining if examining ciliated cells to visualize the axoneme extending from the CEP164-positive centriole

This approach enables precise identification of centriole maturation status in studies of centrosome duplication, cell cycle progression, and ciliogenesis.

What controls should be included when validating a new CEP164 antibody?

Thorough validation of a new CEP164 antibody requires multiple controls to ensure specificity and reliability:

• Positive cellular controls:

  • Cell lines known to express CEP164 strongly: HEK-293, HeLa, A549, PC-3, and hTERT-RPE1 cells

  • For tissue sections: human colon tissue and human cervical cancer tissue have been validated

• Negative controls:

  • Preimmune serum (for polyclonal antibodies) which should show no specific reactivity

  • Secondary antibody only controls to identify non-specific binding

  • CEP164 knockout or knockdown samples (siRNA-depleted cells have been shown to abolish immunoreactivity)

• Specificity controls:

  • Peptide competition assays using the immunizing peptide

  • Western blot analysis to confirm the antibody detects a band of appropriate size (~200 kDa)

  • Cross-validation with a different CEP164 antibody targeting a distinct epitope

• Application-specific controls:

  • For IF: Compare with previously characterized CEP164 staining patterns (ringlike structure at mother centriole)

  • For WB: Compare with exogenously expressed tagged CEP164 (myc-tagged CEP164 has been shown to exhibit similar migration patterns to endogenous protein)

  • For IHC: Include isotype control antibodies

• Cross-reactivity assessment:

  • If studying non-human samples, validate specificity in the relevant species

  • Sequence alignment analysis can predict potential cross-reactivity

Documentation of these validation steps provides essential quality assurance for subsequent experiments and publications.

How do I troubleshoot weak or non-specific CEP164 antibody staining in immunofluorescence?

Troubleshooting CEP164 immunofluorescence staining involves systematic evaluation of multiple factors:

• Fixation optimization:

  • Test different fixation methods (paraformaldehyde vs. methanol)

  • Methanol fixation (5-10 minutes at -20°C) often works better for centrosomal proteins

  • For some epitopes, a combined approach of brief paraformaldehyde fixation followed by methanol may improve results

• Permeabilization considerations:

  • Ensure adequate permeabilization (0.1-0.5% Triton X-100)

  • Extend permeabilization time if nuclear or centrosomal staining is weak

  • For difficult epitopes, try alternative detergents like saponin

• Antigen retrieval methods:

  • For tissue sections, appropriate antigen retrieval is critical

  • For CEP164, TE buffer pH 9.0 or citrate buffer pH 6.0 are recommended

• Antibody dilution optimization:

  • Test a range of dilutions (1:200-1:800 is the recommended range)

  • Extended primary antibody incubation (overnight at 4°C) may improve signal

• Signal amplification strategies:

  • Consider using a biotin-streptavidin system for signal enhancement

  • Tyramide signal amplification can dramatically increase sensitivity

• Background reduction:

  • Increase blocking time and concentration (5% BSA or 10% normal serum)

  • Add 0.1-0.3% Triton X-100 to antibody dilution buffers

  • Include 0.1% Tween-20 in wash buffers and increase washing steps

• Microscopy considerations:

  • CEP164 localizes to a small structure; high-resolution imaging is essential

  • Consider deconvolution or super-resolution techniques for optimal visualization

  • Z-stack imaging helps ensure capture of the centriole plane

If non-specific nuclear staining occurs, pre-absorption of the antibody with nuclear extracts can sometimes improve specificity.

How can CEP164 antibodies be employed in studies of ciliopathies?

CEP164 antibodies provide valuable tools for investigating ciliopathies, as CEP164 dysfunction has been implicated in disorders such as Bardet-Biedl syndrome and Meckel-Gruber syndrome . Implementing these antibodies effectively requires:

• Patient sample analysis approaches:

  • Immunohistochemistry of patient biopsies to assess CEP164 localization abnormalities

  • Quantification of CEP164 expression levels in patient-derived cells

  • Comparative analysis of centriole structure and ciliogenesis capacity using CEP164 as a marker

  • Examination of CEP164 post-translational modifications that might be altered in disease states

• Disease modeling experimental design:

  • Use CEP164 antibodies to validate disease models (patient-derived cells, CRISPR-edited cell lines, animal models)

  • Quantitative assessment of:

    • Percentage of cells with CEP164-positive centrioles

    • Intensity and morphology of CEP164 staining at distal appendages

    • Correlation between CEP164 localization and primary cilia formation

• Pathway analysis methodologies:

  • Co-immunoprecipitation with CEP164 antibodies to identify altered protein interactions in disease states

  • Combined CEP164 immunostaining with markers of ciliary trafficking or signaling pathways

  • Correlative analysis of CEP164 localization with ciliopathy phenotypes

• Therapeutic screening applications:

  • Using CEP164 immunostaining as a readout for high-content screening of compounds that might restore normal centriole function

  • Monitoring CEP164 localization and function during gene therapy approaches

• Implementation considerations:

  • Different tissue types may require optimized protocols

  • Control samples from healthy individuals must be processed identically

  • Quantitative image analysis using standardized parameters enhances reproducibility

This approach enables researchers to determine whether ciliopathy phenotypes correlate with specific defects in CEP164 localization, expression, or function, potentially revealing novel therapeutic targets.

What are the methodological considerations for studying CEP164 phosphorylation during cell cycle progression?

Investigating CEP164 phosphorylation states requires specialized approaches:

• Phospho-specific antibody development and validation:

  • While general CEP164 antibodies detect the protein regardless of phosphorylation state, phospho-specific antibodies may need to be custom-developed

  • Validation should include:

    • Western blot comparison before and after phosphatase treatment

    • Mutation of putative phosphorylation sites (alanine substitution)

    • Mass spectrometry confirmation of specificity

• Cell synchronization protocols:

  • For G1/S boundary: Double thymidine block

  • For G2/M: Nocodazole treatment

  • For mitotic stages: Release from nocodazole with time-course analysis

  • Confirmation of synchronization using flow cytometry or specific cell cycle markers

• Kinase inhibition experiments:

  • Treatment with specific kinase inhibitors to identify regulatory kinases

  • Analysis of CEP164 phosphorylation status following inhibitor treatment

  • Correlation with functional outcomes (centriole maturation, ciliogenesis capability)

• Combining techniques for comprehensive analysis:

  • Immunoprecipitation with general CEP164 antibodies followed by phospho-specific Western blotting

  • Immunofluorescence using phospho-specific antibodies to determine localization changes

  • Phos-tag gel electrophoresis to separate differentially phosphorylated forms

• Analysis framework:

  • Quantitative Western blot analysis normalizing phospho-signal to total CEP164

  • Time-course experiments throughout cell cycle with multiple timepoints

  • Correlation of phosphorylation changes with functional outcomes

This methodology allows researchers to connect CEP164 phosphorylation events with specific cell cycle transitions and centrosomal functions, revealing regulatory mechanisms governing centriole maturation and primary cilium formation.

How can super-resolution microscopy enhance CEP164 antibody applications?

Super-resolution microscopy significantly enhances CEP164 visualization, revealing details impossible to resolve with conventional microscopy:

• Technique selection considerations:

  • Structured Illumination Microscopy (SIM): Provides ~100 nm resolution, revealing the ring-like arrangement of CEP164 at distal appendages

  • Stimulated Emission Depletion (STED): Achieves ~20-50 nm resolution, allowing visualization of individual distal appendage substructures

  • Single Molecule Localization Microscopy (STORM/PALM): Offers ~10-20 nm resolution, enabling precise positioning of CEP164 relative to other proteins

• Sample preparation optimizations:

  • Minimize sample thickness: Grow cells on high-precision coverslips

  • Reduce background: Use highly specific primary antibodies and minimize non-specific binding

  • Implement specialized fixation: Glutaraldehyde addition (0.1-0.2%) can help preserve ultrastructure

  • Select appropriate fluorophores: Choose bright, photostable dyes compatible with super-resolution techniques

• Multi-color experimental design:

  • Carefully select fluorophore combinations to avoid bleed-through

  • Include reference markers such as γ-tubulin (centriole barrel) and acetylated tubulin (ciliary axoneme)

  • Consider sequential staining approaches for difficult combinations

• Data analysis approaches:

  • 3D reconstruction to visualize the complete distal appendage structure

  • Quantitative analysis of CEP164 distribution patterns

  • Nearest neighbor analysis to determine spatial relationships with other proteins

  • Temporal analysis in live-cell experiments (if applicable)

• Expected insights:

  • Precise mapping of CEP164 within the ninefold symmetrical arrangement of distal appendages

  • Detailed visualization of structural changes during centriole maturation

  • Nanoscale distribution changes in response to cellular signals or in disease models

These approaches have revealed that CEP164 forms distinct "arms" at the distal appendages rather than a continuous ring, information that was not distinguishable using conventional microscopy techniques.

What experimental design best investigates CEP164's role in the DNA damage response?

Recent research has connected CEP164 to DNA damage response pathways. An optimal experimental design to investigate this connection includes:

• Integrated analysis approach:

  • Create stable cell lines with inducible CEP164 knockdown or overexpression

  • Establish complementary systems with CEP164 mutants lacking specific domains

  • Develop reporter cell lines to simultaneously monitor CEP164 status and DNA damage markers

• Damage induction protocols:

  • Compare different DNA damaging agents (ionizing radiation, UV, chemical agents)

  • Apply dose-response analysis to identify threshold effects

  • Use micro-irradiation to induce localized damage for real-time recruitment studies

• Multi-parameter assessment:

  • Immunofluorescence co-localization studies of CEP164 with DNA damage markers (γH2AX, 53BP1)

  • Western blot analysis of CEP164 modifications following DNA damage

  • Chromatin immunoprecipitation to assess CEP164 association with damaged DNA regions

  • Flow cytometry analysis of cell cycle progression and checkpoint activation

• Functional outcome measurements:

  • Comet assay to assess DNA break repair kinetics

  • Survival assays to determine functional consequences of CEP164 manipulation

  • Cell cycle analysis to identify specific checkpoint dependencies

  • Assessment of genomic stability markers (micronuclei formation, chromosomal aberrations)

• Mechanistic dissection:

  • Proximity labeling approaches to identify damage-specific interaction partners

  • Domain deletion analysis to map regions required for damage response

  • Phospho-proteomic analysis to identify damage-induced modifications

  • RNA-seq to assess transcriptional consequences of CEP164 dysfunction during damage response

This comprehensive approach enables researchers to distinguish CEP164's centrosomal functions from potential direct roles in DNA damage signaling or repair pathways, clarifying this protein's multifunctional nature.

What emerging technologies are expanding CEP164 antibody applications?

Several cutting-edge technologies are enhancing the research applications of CEP164 antibodies:

• Single-cell protein analysis:

  • Mass cytometry (CyTOF) incorporating CEP164 antibodies allows simultaneous analysis of centrosomal status and multiple cellular parameters at single-cell resolution

  • Microfluidic approaches for temporal analysis of CEP164 dynamics in individual cells

• Proximity labeling approaches:

  • BioID or APEX2 fusions with CEP164 enable identification of proximal proteins in living cells

  • Spatial proteomics revealing the CEP164 "interactome" at different cell cycle stages

  • TurboID variants for rapid labeling of transient interactions

• Live-cell imaging innovations:

  • Development of intrabodies or nanobodies against CEP164 for live-cell applications

  • Split-fluorescent protein complementation assays to visualize CEP164 interactions in real-time

  • Lattice light-sheet microscopy for extended, high-resolution imaging of CEP164 dynamics

• Spatial transcriptomics integration:

  • Combining CEP164 immunostaining with spatial transcriptomics to correlate centrosome status with local gene expression patterns

  • RNA-protein co-detection methods to identify RNAs associated with CEP164-positive structures

These emerging methods are expanding our understanding of CEP164 beyond static localization to dynamic functional relationships in diverse cellular contexts, offering new insights into centrosome biology and ciliopathies.