CEP126 Antibody

What is CEP126 and why is it important in cellular research?

CEP126 is a centrosomal protein that localizes to the centrosome, pericentriolar satellites, and the base of the primary cilium. It plays crucial roles in:

• Regulation of microtubule organization at the centrosome

• Pericentriolar satellite transport to the centrosome

• Primary cilium formation

• Mitotic spindle organization

• Cytokinesis

Research has demonstrated that CEP126 depletion results in dispersion of pericentriolar satellites and disruption of the radial organization of microtubules. Additionally, it causes disorganization of the mitotic spindle and impairs primary cilium formation in hTERT-RPE-1 and IMCD3 cells .

What detection methods are available for CEP126 antibodies?

CEP126 antibodies can be detected through multiple techniques:

For optimal results, researchers should validate antibodies for their specific application and cell type. When using immunofluorescence, co-staining with centrosomal markers like γ-tubulin is recommended for accurate identification of centrosomal localization .

What are the key considerations for selecting a CEP126 antibody?

When selecting a CEP126 antibody, consider:

• Specificity: Confirm the antibody specifically recognizes CEP126 with minimal cross-reactivity

• Host species: Consider compatibility with other antibodies for co-localization studies

• Application validation: Ensure the antibody is validated for your specific application (WB, IF, IHC, ELISA)

• Epitope recognition: Choose antibodies recognizing functionally relevant domains based on your research questions

• Clonality: Polyclonal antibodies may recognize multiple epitopes, while monoclonal antibodies offer higher specificity

For critical experiments, validation through siRNA knockdown controls is recommended to confirm specificity and sensitivity of the selected antibody .

How can I visualize CEP126 localization in cells?

To visualize CEP126 localization:

• Fix cells with 4% paraformaldehyde (10 minutes) or ice-cold methanol (5 minutes) depending on epitope accessibility

• Permeabilize with 0.2% Triton X-100 if using paraformaldehyde fixation

• Block with 3-5% BSA or normal serum from the secondary antibody host species

• Incubate with anti-CEP126 antibody (typically 1:100-1:500 dilution)

• Counterstain with markers for:

  • Centrosome (γ-tubulin)

  • Cilia (acetylated tubulin)

  • Pericentriolar satellites (PCM1)

• Use confocal microscopy for best resolution of centrosomal and ciliary structures

Live imaging can also be performed with transfection of fluorescently tagged CEP126, though caution should be taken as overexpression may affect normal protein function .

How can I study the functional domains of CEP126 in cellular processes?

To investigate CEP126 functional domains:

• Expression of deletion mutants: Create constructs lacking specific domains (e.g., C-terminal domain, centrosome localization domain)

  • The 1-967 truncation mutant (lacking C-terminal domain) localizes to the centrosome but severely impairs microtubule organization

  • The 520-655 region contains the putative centrosome-localization domain

• Domain-specific antibodies: Use antibodies recognizing specific domains to study localization patterns

• Point mutations: Introduce specific mutations in functional domains to study their effects

• Rescue experiments: Deplete endogenous CEP126 using siRNA and express RNAi-resistant wildtype or mutant CEP126 to determine which domains are essential for function

Research has shown that expression of the 1-967 CEP126 truncation mutant caused >80% reduction in ciliated cells, demonstrating the importance of the C-terminal domain in cilium formation .

What are the optimal experimental designs for studying CEP126's role in primary cilium formation?

To study CEP126's role in cilium formation:

• Cell models: Use cilium-forming cell lines like hTERT-RPE-1 or IMCD3

• Cilium induction protocol:

  • Grow cells to 70-80% confluence

  • Serum-starve for 24-48 hours to induce cilium formation

  • Fix and stain with anti-acetylated tubulin (cilium marker) and anti-CEP126

• Quantification metrics:

  • Percentage of ciliated cells

  • Cilium length

  • CEP126 localization at cilium base

• Perturbation approaches:

  • siRNA knockdown (two different siRNAs to control for off-target effects)

  • Expression of truncation mutants (particularly the 1-967 mutant)

  • CRISPR/Cas9 knockout (for long-term studies)

• Rescue experiments:

  • Express RNAi-resistant wildtype CEP126 in knockdown cells

Studies have demonstrated that CEP126 depletion reduced the percentage of ciliated cells by >80%, and similar results were observed with expression of the 1-967 truncation mutant, highlighting CEP126's essential role in cilium formation .

What protocols are recommended for studying interactions between CEP126 and other centrosomal proteins?

To investigate CEP126 protein interactions:

• Co-immunoprecipitation:

  • Transfect cells with tagged CEP126 (e.g., Flag-tagged)

  • Lyse cells in buffer containing 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 1% NP-40, and protease inhibitors

  • Immunoprecipitate with anti-tag antibody

  • Analyze by Western blot for interacting proteins

• Proximity ligation assay (PLA):

  • Allows detection of protein interactions in situ

  • Requires antibodies from different species against CEP126 and potential interactors

• Yeast two-hybrid screening:

  • Use CEP126 domains as bait to identify novel interactors

• Mass spectrometry:

  • Immunoprecipitate CEP126 and perform mass spectrometry analysis

  • Compare results from different cell types or conditions

CEP126 has been shown to interact with p150Glued, a subunit of the dynein-dynactin complex, which is involved in the transport of pericentriolar satellites. Both full-length CEP126 and the 1-967 truncation mutant interact with p150Glued .

How can I quantitatively assess CEP126 expression levels in experimental samples?

For quantitative assessment of CEP126:

• Western blot quantification:

  • Use β-actin or GAPDH as loading controls

  • Perform densitometry analysis with software like ImageJ

  • Run multiple biological replicates for statistical significance

• ELISA:

  • Commercial CEP126 ELISA kits are available with detection ranges of 7.813-500 ng/mL

  • Minimum detection limit: approximately 4.688-7.813 ng/mL

  • Sample types: serum, plasma, tissue homogenates

• RT-qPCR:

  • Design primers spanning exon-exon junctions

  • Normalize to housekeeping genes

  • Validate knockdown efficiency at the mRNA level

• Immunofluorescence quantification:

  • Measure fluorescence intensity at the centrosome

  • Use automated image analysis software for unbiased quantification

  • Compare to control conditions with consistent imaging parameters

When using ELISA for CEP126 detection, sample dilution (at least 1:2 with Sample Dilution Buffer) is recommended for optimal results .

What are the technical challenges in studying CEP126's role in microtubule organization and how can they be addressed?

Challenges and solutions for studying CEP126's role in microtubule organization:

• Dynamic nature of microtubules:

  • Use live imaging with EB1-GFP to track growing microtubule plus ends

  • Perform microtubule regrowth assays after nocodazole treatment

• Distinguishing direct vs. indirect effects:

  • Compare acute (siRNA) vs. chronic (CRISPR) depletion methods

  • Use rescue experiments with domain mutants

• Visualization of microtubule networks:

  • Use COS7 cells, which have well-defined astral microtubule arrays

  • Employ super-resolution microscopy techniques

• Quantification challenges:

  • Develop automated image analysis methods to quantify:

    • Microtubule radial organization

    • Centrosomal microtubule density

    • Microtubule dynamics parameters

• Distinguishing CEP126 functions:

  • Use carefully timed expression of mutants (e.g., 20-hour expression of the 1-967 truncation mutant)

  • Combine with cell cycle synchronization methods

Research has shown that in CEP126-depleted cells, microtubules do not radiate from the centrosome, with most microtubules randomly oriented throughout the cytoplasm. Less than 20% of CEP126 knockdown cells showed a focused microtubule array compared to more than 80% of control cells .

How do antibody-based detection methods for CEP126 compare with other molecular techniques?

Comparison of detection methods:

For the most comprehensive understanding of CEP126 biology, combining multiple approaches is recommended. For example, validating antibody specificity by comparing with tagged protein expression and confirming knockdown by both qPCR and Western blot .

What approaches can be used to study CEP126's interaction with pericentriolar satellites?

To study CEP126's role in pericentriolar satellite function:

• Co-localization studies:

  • Co-stain for CEP126 and pericentriolar satellite markers (PCM1, pericentrin)

  • Use dual-color live imaging with tagged proteins

  • Apply super-resolution microscopy techniques

• Functional assays:

  • Measure pericentriolar satellite motility using live imaging

  • Assess satellite distribution after CEP126 depletion

  • Quantify pericentrin and PCM1 dispersion

• Microtubule dependency:

  • Treat with nocodazole to disrupt microtubules

  • Examine satellite redistribution in CEP126 normal vs. depleted cells

• Motor protein involvement:

  • Investigate the interaction between CEP126 and p150Glued

  • Use dynein inhibitors to compare with CEP126 depletion effects

  • Perform co-immunoprecipitation of CEP126 with motor proteins

Research has shown that in CEP126-depleted cells, PCM1-positive pericentriolar satellites become dispersed throughout the cytoplasm rather than clustering around the centrosome. This phenotype resembles what occurs when dynein-dynactin function is disrupted, consistent with CEP126's interaction with p150Glued .

What is the relationship between CEP126 and cell cycle progression, and how can this be experimentally addressed?

To study CEP126's role in cell cycle:

• Cell synchronization approaches:

  • Double thymidine block for G1/S synchronization

  • Nocodazole block and release for M-phase studies

  • Serum starvation/stimulation for G0/G1 transition

• Cell cycle markers:

  • Co-stain CEP126 with:

    • Cyclin B1 (G2/M)

    • PCNA (S phase)

    • Phospho-histone H3 (mitosis)

• Live cell cycle reporters:

  • FUCCI system to track cell cycle progression

  • Combine with CEP126-FP fusions

• Functional assays:

  • Flow cytometry analysis of cell cycle distribution after CEP126 depletion

  • Time-lapse imaging of mitotic progression

  • Spindle assembly checkpoint activation assessment

• Mitotic defect analysis:

  • Quantify spindle abnormalities in CEP126-depleted cells

  • Measure mitotic duration

  • Assess cytokinesis completion rates

Research has demonstrated that CEP126 localizes to the centrosome throughout the cell cycle and to the midbody during cytokinesis. CEP126 depletion induces disorganization of the mitotic spindle, suggesting it plays important roles in mitotic progression .

How can I troubleshoot inconsistent CEP126 antibody staining patterns?

Common issues and solutions:

• High background staining:

  • Increase blocking time/concentration (5% BSA, 1 hour)

  • Reduce primary antibody concentration

  • Include 0.1-0.3% Triton X-100 in antibody dilution buffers

  • Use more stringent washing (0.1% Tween-20 in PBS, 3×10 minutes)

• No centrosomal signal:

  • Try different fixation methods (PFA vs. methanol)

  • Optimize antigen retrieval (if applicable)

  • Confirm antibody recognizes native protein conformation

  • Verify epitope accessibility in your experimental conditions

• Variable staining intensity:

  • Standardize cell culture conditions

  • Control for cell cycle stage (synchronized populations)

  • Optimize antibody concentration using titration experiments

  • Use consistent imaging parameters

• Non-centrosomal staining:

  • Validate antibody specificity using siRNA knockdown

  • Use multiple antibodies targeting different epitopes

  • Include appropriate negative controls

• Cell type variation:

  • Optimize protocols for each cell line

  • Consider expression levels in different cell types

  • Adjust antibody concentration accordingly

Researchers have reported that while CEP126 clearly localizes to the centrosome, the pericentriolar satellite localization is more readily visualized using overexpressed tagged protein rather than by immunolabeling of endogenous CEP126 in fixed cells .

How do I interpret contradictory results when studying CEP126 function?

When facing contradictory results:

• Methodological differences:

  • Compare experimental approaches (transient vs. stable knockdown)

  • Evaluate knockdown efficiency across studies

  • Consider cell-type specific differences

  • Examine timing of analyses (acute vs. chronic effects)

• Antibody considerations:

  • Verify antibodies recognize the same epitopes

  • Confirm specificity in your experimental system

  • Use multiple antibodies when possible

• Functional redundancy:

  • Consider compensation by related proteins

  • Investigate potential adaptation in long-term knockdowns

  • Perform double knockdowns of functionally related proteins

• Cell cycle effects:

  • Synchronize cells to eliminate cell-cycle variability

  • Analyze phenotypes in specific cell cycle stages

• Technical validation:

  • Include proper controls (positive, negative, and technical)

  • Use complementary approaches (fixed and live imaging)

  • Quantify results with appropriate statistical analysis

The complex nature of centrosome biology often results in seemingly contradictory findings. For example, different truncation mutants of CEP126 may show varying effects on microtubule organization depending on the domains affected and expression levels .

What data analysis approaches are recommended for quantifying CEP126-related phenotypes?

For robust quantification:

• Automated image analysis workflows:

  • Use CellProfiler or ImageJ/Fiji for unbiased quantification

  • Develop macros for batch processing of images

  • Implement machine learning approaches for complex phenotypes

• Quantification metrics for common phenotypes:

  Phenotype	Recommended Metrics	Analysis Approach
  Satellite dispersion	Distance from centrosome	Radial distribution analysis
  MT organization	Angle variance from centrosome	Radial line scan analysis
  Cilium formation	% ciliated cells, cilium length	Automated detection and measurement
  Protein localization	Intensity at centrosome vs. cytoplasm	Ratio analysis with defined ROIs
  Spindle defects	Spindle angle, pole distance	3D measurement tools

• Statistical considerations:

  • Use appropriate statistical tests (t-test, ANOVA)

  • Account for multiple comparisons

  • Report effect sizes along with p-values

  • Include biological replicates (n≥3)

• Data presentation:

  • Include representative images alongside quantification

  • Use consistent scaling and color schemes

  • Provide clear legends and methodology descriptions

For robust analysis of microtubule organization defects, researchers have quantified the percentage of cells with focused microtubule arrays, finding that while >80% of control cells showed focused arrays, <20% of CEP126 knockdown cells maintained this organization .

How might single-cell approaches enhance our understanding of CEP126 function?

Single-cell methodologies offer several advantages:

• Single-cell RNA sequencing:

  • Reveal cell-to-cell variation in CEP126 expression

  • Identify co-regulated gene networks

  • Discover cell-type specific expression patterns

  • Track changes across cell cycle or differentiation

• Single-cell protein analysis:

  • Mass cytometry (CyTOF) for multi-parameter protein analysis

  • Single-cell Western blotting for protein heterogeneity

  • Correlate CEP126 levels with other centrosomal proteins

• Live single-cell imaging:

  • Track dynamic changes in CEP126 localization

  • Measure protein turnover rates using photobleaching

  • Correlate phenotypes with expression levels

  • Examine cell-to-cell variability in response to perturbations

• CRISPR screens at single-cell resolution:

  • Identify genetic interactions with CEP126

  • Discover context-dependent functions

  • Link genotype to phenotype at single-cell level

These approaches could reveal how CEP126 expression heterogeneity impacts centrosome function and cilium formation across different cell populations or within developing tissues .

What are the newest methodologies for studying CEP126 antibody specificity and cross-reactivity?

Advanced approaches for antibody validation:

• CRISPR knockout validation:

  • Generate CEP126 knockout cell lines as ultimate negative controls

  • Perform Western blot and immunofluorescence to confirm antibody specificity

  • Create epitope-deleted cell lines for domain-specific antibodies

• Orthogonal antibody testing:

  • Compare antibodies recognizing different epitopes

  • Correlate results from different detection methods

  • Use tagged proteins as reference standards

• Mass spectrometry validation:

  • Identify all proteins pulled down by CEP126 antibodies

  • Quantify on-target vs. off-target binding

  • Map precise epitopes recognized by the antibody

• High-throughput antibody screening:

  • Test multiple antibodies against protein arrays

  • Evaluate cross-reactivity profiles systematically

  • Develop antibody specificity scores

• In situ antibody validation:

  • Proximity ligation assays with multiple antibody pairs

  • Pre-absorption controls with recombinant antigens

  • Competitive binding assays

For CEP126 antibodies, researchers should be particularly vigilant about cross-reactivity with other centrosomal proteins, as the centrosome contains hundreds of proteins in a small volume .

How can high-throughput screening approaches be applied to identify modulators of CEP126 function?

High-throughput screening strategies:

• Genetic screens:

  • CRISPR/Cas9 knockout libraries targeting the genome

  • siRNA/shRNA libraries for transient knockdown

  • cDNA overexpression libraries to identify suppressors

• Screening readouts:

  • High-content imaging for cilium formation

  • Automated microtubule organization analysis

  • Reporter assays for centrosome-dependent processes

• Chemical screens:

  • Small molecule libraries targeting kinases, phosphatases

  • Compounds affecting microtubule dynamics

  • Targeted degraders (PROTACs) for acute protein depletion

• Biosensor approaches:

  • FRET sensors for protein-protein interactions

  • Split-GFP complementation for proximity detection

  • Activity-based probes for functional readouts

• Data integration:

  • Combine genetic and chemical screening data

  • Apply machine learning for pattern recognition

  • Integrate with public datasets for broader context

High-throughput approaches could be particularly valuable for identifying factors that regulate CEP126's interactions with the dynein-dynactin complex or proteins involved in pericentriolar satellite transport .

What role might CEP126 play in ciliopathies and centrosome-related diseases?

Potential disease connections for CEP126:

• Primary ciliopathies:

  • Given CEP126's role in cilium formation, mutations could contribute to:

    • Polycystic kidney disease

    • Bardet-Biedl syndrome

    • Joubert syndrome

    • Primary ciliary dyskinesia

• Centrosome amplification in cancer:

  • CEP126 dysregulation might affect:

    • Centrosome duplication control

    • Mitotic spindle organization

    • Genomic stability maintenance

• Microcephaly and neurodevelopmental disorders:

  • Proper centrosome function is critical for neurogenesis

  • CEP126 mutations could affect neural progenitor division

  • Brain development might be particularly sensitive to CEP126 dysfunction

• Experimental approaches to investigate disease connections:

  • Patient-derived cell studies

  • CRISPR engineering of disease-associated mutations

  • Animal models of CEP126 dysfunction

  • Tissue-specific knockouts to assess organ-specific effects

The essential role of CEP126 in cilium formation suggests it could be involved in ciliopathies, though specific disease associations have not yet been definitively established in the literature .

How can advanced imaging techniques enhance our understanding of CEP126 dynamics?

Cutting-edge imaging approaches:

• Super-resolution microscopy:

  • STED, STORM, or PALM imaging for nanoscale localization

  • Resolve subdomains within centrosomes (~200-500 nm)

  • Map precise CEP126 localization relative to centrioles

• Live-cell super-resolution:

  • Track dynamic movement of CEP126

  • Observe pericentriolar satellite transport in real-time

  • Correlate with microtubule dynamics

• Correlative light and electron microscopy (CLEM):

  • Combine fluorescence with ultrastructural details

  • Link CEP126 localization to specific centrosomal structures

  • Examine cilium base architecture

• Light-sheet microscopy:

  • Reduced phototoxicity for long-term imaging

  • Track CEP126 dynamics through entire cell cycle

  • Observe cilium formation in real-time

• Expansion microscopy:

  • Physical expansion of specimens for improved resolution

  • Visualize protein arrangements within centrosome

  • Compatible with standard confocal microscopy

Advanced imaging could reveal how CEP126 participates in the transport of pericentriolar satellites and interacts with the dynein-dynactin complex, which is challenging to observe with conventional microscopy .