CEP135 Antibody, FITC conjugated

What is CEP135 and what is its cellular localization pattern?

CEP135 is a 135 kDa centrosomal protein present in a wide range of organisms from sea urchins to mammals. It localizes to the centrosome throughout the cell cycle, and its localization is independent of the microtubule network as demonstrated by nocodazole treatment experiments . Immunoelectron microscopy reveals that CEP135 distributes throughout the centrosomal area in association with the electron-dense material surrounding centrioles . This protein is considered a universal component of centrosomes, with homologues ranging from 120 to 140 kDa detected across various species including Xenopus, sea urchin, and numerous mammalian cell types .

What are the key structural features of the CEP135 protein?

CEP135 is a protein of 1,145 amino acid residues with extensive α-helical domains favoring coiled-coil structure formation . Sequence analysis of cDNA isolated from CHO cells predicted this structure, which is consistent with its role in centrosomal organization. The protein contains at least three independent centrosomal targeting domains that span almost the entire length of the CEP135 sequence, allowing it to properly localize to the centrosome . These structural characteristics enable CEP135 to function as a scaffolding protein during early centriole biogenesis and to serve as a platform for other centrosomal proteins like CEP250 .

What is the functional significance of CEP135 in centrosome biology?

CEP135 plays crucial roles in:

• Centriole biogenesis - Acting as a scaffolding protein during early stages of centriole formation

• Centriole-centriole cohesion - Functioning as a platform protein for CEP250 at the centriole during interphase

• Microtubule organization - Altered levels of CEP135 (through overexpression or RNAi) significantly affect microtubule patterns in cells

• Centrosome stability - Maintains centrosomal integrity as evidenced by the severe consequences of its mutation

Studies using RNAi to suppress CEP135 expression have shown that reducing CEP135 levels by 85-90% significantly impacts microtubule organization, confirming its importance in centrosome function .

What are the optimal conditions for using FITC-conjugated anti-CEP135 antibodies in immunofluorescence?

When performing immunofluorescence with FITC-conjugated anti-CEP135 antibodies, it's critical to include proper controls and to optimize fixation conditions. Wild-type CEP135 localizes specifically to centrosomes and co-localizes with other centrosomal markers like pericentrin, which should be used to validate staining patterns . Researchers should be aware that overexpression of CEP135 can lead to abnormal microtubule networks and multiple centrosomes (up to 5 per cell), which may complicate interpretation .

How should researchers validate the specificity of CEP135 FITC-conjugated antibodies?

A multi-tiered validation approach is recommended:

• Co-localization studies: Confirm that the FITC-CEP135 signal co-localizes with established centrosomal markers such as pericentrin or γ-tubulin .

• Sibling antibody comparison: Compare staining patterns with other validated CEP135 antibodies from different hosts or clones.

• Genetic controls: Use cells with known CEP135 knockdown/knockout status. RNAi experiments have shown that reducing CEP135 expression by 85-90% results in correspondingly reduced centrosomal staining .

• Expression system controls: Compare staining in cells transfected with wild-type versus mutant CEP135 (e.g., the frameshift mutation c.970delC which causes protein truncation) .

• Western blot correlation: Confirm that antibody detects the expected ~135 kDa band in Western blots of whole cell lysates and isolated mitotic spindle preparations .

What methodological considerations are important when studying CEP135 mutations?

When investigating CEP135 mutations:

• RNA analysis: Design appropriate RT-PCR protocols to detect potential nonsense-mediated mRNA decay. In the case of the c.970delC mutation, RT-PCR revealed that the mutant mRNA was degraded, as no PCR product was obtained from patient samples while control samples yielded the expected ~3.4 kb product .

• Protein localization: When expressing wild-type versus mutant CEP135, note that while wild-type GFP-tagged CEP135 localizes to the centrosome and co-localizes with pericentrin, mutant forms may show diffuse cytoplasmic staining with no centrosomal localization .

• Phenotypic analysis: Monitor for multiple centrosomes (up to 5) and multipolar spindle formation, which are observed in cells expressing mutant but not wild-type CEP135 .

• Microtubule organization: Assess microtubule network organization, as both wild-type and mutant CEP135 overexpression can lead to abnormal patterns, with more severe disorganization in cells expressing mutant protein .

How can researchers utilize CEP135 antibodies to study primary microcephaly?

Primary microcephaly has been linked to mutations in CEP135, including the frameshift mutation c.970delC (p.Gln324Serfs∗2) which causes protein truncation . Researchers can:

• Genetic screening: Use sequencing to identify CEP135 mutations in microcephaly patients, particularly focusing on regions showing homozygosity in affected families.

• Functional characterization: Express wild-type and mutant CEP135 tagged with fluorescent reporters to compare centrosomal localization and function.

• Cellular phenotyping: Analyze patient-derived fibroblasts for centrosomal abnormalities and microtubule organization defects.

• Rescue experiments: Determine if reintroducing wild-type CEP135 can rescue cellular phenotypes in patient-derived cells.

• Brain organoid models: Generate cerebral organoids from patient-derived iPSCs to study the impact of CEP135 mutations on brain development in a three-dimensional context.

When using FITC-conjugated antibodies in these studies, researchers should carefully titrate antibody concentrations and include appropriate controls to distinguish between specific signal and background fluorescence .

What approaches can be used to investigate CEP135 in centrosome amplification mechanisms?

Centrosome amplification is linked to genomic instability and cancer progression. To study CEP135's role:

• Cell cycle synchronization: Synchronize cells and analyze CEP135 expression and localization at different cell cycle stages.

• Quantitative imaging: Develop high-content screening approaches using FITC-CEP135 antibodies to quantify centrosome number, size, and CEP135 intensity.

• Cancer cell models: Compare CEP135 expression and localization in normal versus cancer cell lines with known centrosome amplification.

• Isoform analysis: Investigate CEP135 isoform dysregulation as it has been linked to centrosome amplification .

• Drug response studies: Monitor CEP135 localization following treatment with drugs that affect centrosome duplication or microtubule dynamics.

For these applications, a FITC-conjugated antibody dilution of 1:200-1:800 for immunofluorescence is typically suitable, though optimization for specific cell types is recommended .

How can researchers investigate the interaction between CEP135 and other centrosomal proteins?

To study CEP135 protein interactions:

• Co-immunoprecipitation: Use CEP135 antibodies to pull down protein complexes, followed by mass spectrometry or Western blot analysis to identify interacting partners.

• Proximity ligation assays: Combine CEP135 FITC-conjugated antibodies with antibodies against suspected interacting proteins to visualize protein-protein interactions in situ.

• FRET/FLIM analysis: Express CEP135 fused to a FRET donor and potential interacting proteins fused to acceptor fluorophores.

• Yeast two-hybrid screening: Identify novel interacting partners using CEP135 as bait.

• Domain mapping: Express truncated versions of CEP135 containing specific domains to determine which regions mediate interactions with other proteins, similar to the approach used to identify the three independent centrosomal targeting domains .

When performing co-localization studies, it's important to note that while CEP135 localizes to the centrosome, it has also been detected in some spots outside the centrosome that were not positive for pericentrin .

What factors might affect CEP135 detection using FITC-conjugated antibodies?

When troubleshooting CEP135 detection issues, it's important to note that mutant CEP135 may not localize to the centrosome at all but instead show diffuse cytoplasmic staining in a small percentage of cells .

How should researchers interpret CEP135 localization patterns in the context of centrosome abnormalities?

Interpretation guidelines:

• Normal pattern: In healthy cells, CEP135 appears as 1-2 distinct foci that co-localize with other centrosomal markers like pericentrin and γ-tubulin .

• Supernumerary foci: Multiple CEP135-positive foci (>2 per cell) may indicate centrosome amplification, which could result from CEP135 overexpression, mutation, or dysregulation of centrosome duplication .

• Diffuse staining: Diffuse cytoplasmic distribution rather than focal centrosomal localization often indicates mutant protein expression or disruption of centrosome integrity .

• Fibrillary structures: Overexpression of CEP135 can lead to the formation of unique whorl-like particles in both the centrosome and cytoplasm, composed of parallel dense lines arranged in a 6-nm space .

• Microtubule association: In some overexpression studies, CEP135 has been detected on microtubules, suggesting a potential direct interaction with the microtubule network .

When analyzing these patterns, it's critical to correlate CEP135 localization with functional outcomes such as microtubule organization and mitotic spindle formation.

How can advanced imaging techniques enhance CEP135 research using FITC-conjugated antibodies?

Cutting-edge imaging approaches for CEP135 studies include:

• Super-resolution microscopy: Techniques like STED, SIM, or STORM can resolve centrosomal substructures beyond the diffraction limit, revealing precise CEP135 localization within the centrosome.

• Live-cell imaging: While FITC-conjugated antibodies are typically used for fixed cells, researchers can combine these studies with live-cell imaging of fluorescently-tagged CEP135 to correlate dynamic behavior with fixed-cell observations.

• Correlative light and electron microscopy (CLEM): This approach allows researchers to first identify CEP135-positive structures by fluorescence microscopy and then examine their ultrastructure by electron microscopy, similar to the approach used in initial CEP135 characterization studies .

• High-content screening: Automated imaging platforms can quantify CEP135 parameters across thousands of cells, enabling large-scale studies of genetic or chemical perturbations.

• Expansion microscopy: Physical expansion of specimens can provide enhanced resolution of centrosomal structures using standard fluorescence microscopes.

When employing these advanced techniques, researchers should carefully optimize FITC-conjugated antibody concentrations to achieve the best signal-to-noise ratio for each specific application.

What new directions are emerging in CEP135 research that could benefit from FITC-conjugated antibodies?

Emerging research areas include:

• Single-cell analysis: Investigating cell-to-cell variability in CEP135 expression and localization across tissues and disease states.

• Brain development: Further exploring the role of CEP135 in neurogenesis and microcephaly pathogenesis .

• Cancer biology: Examining the relationship between CEP135 dysregulation, centrosome amplification, and genomic instability in various cancer types .

• Therapeutic targeting: Developing approaches to modulate CEP135 function as a potential therapeutic strategy for centrosome-related disorders.

• Evolutionary conservation: Comparative studies of CEP135 structure and function across species to understand conserved mechanisms of centrosome organization.

FITC-conjugated CEP135 antibodies with recommended dilutions of 1:50-200 for immunofluorescence applications will continue to be valuable tools in these emerging research directions, particularly when combined with other centrosomal markers and advanced imaging technologies.