cep63 Antibody

What is CEP63 and what are its primary cellular functions?

CEP63 is a 63 kDa centrosomal protein with multiple critical cellular functions:

• Required for normal spindle assembly during cell division

• Maintains centrosome numbers through centrosomal recruitment of CEP152

• Recruits CDK1 to centrosomes

• Plays a significant role in DNA damage response pathways

The protein exists in four isoforms with molecular weights of 56 kDa, 58 kDa, 63 kDa, and 81 kDa, though the observed molecular weight typically ranges from 57-75 kDa in experimental conditions . CEP63's centrosomal localization makes it a valuable marker for studying centrosome biology and related cellular processes.

Which CEP63 antibodies are available for research and what are their properties?

Multiple validated CEP63 antibodies are available for research applications:

Both antibodies are purified (antigen affinity or Protein G), stored in PBS with 0.02% sodium azide and 50% glycerol (pH 7.3), and remain stable for one year when stored at -20°C .

What are the recommended dilutions for different applications of CEP63 antibodies?

Optimal dilutions vary by application and specific antibody:

Researchers should note that these are starting recommendations, and optimal dilutions may vary depending on sample type, detection method, and experimental conditions. It is advisable to titrate the antibody in each testing system to obtain optimal results .

Which cell lines and tissues show reliable CEP63 expression for positive controls?

Several validated cell lines and tissues demonstrate consistent CEP63 expression:

For immunofluorescence applications, MDCK cells and hTERT-RPE1 cells have been validated for 16268-1-AP and 66996-1-Ig antibodies, respectively . These cell lines provide reliable positive controls for antibody validation and experimental optimization.

How can researchers study the interaction between CEP63 and CEP152?

The CEP63-CEP152 interaction is critical for centriole duplication and can be investigated through several approaches:

• Co-immunoprecipitation (Co-IP):

  • Flag-tagged CEP63 efficiently pulls down endogenous CEP152

  • The C-terminus of CEP63 (specifically residues 425-541) is sufficient for this interaction

• Pull-down assays:

  • MBP-tagged CEP63 can be used with amylose resin (incubate 2 μg MBP-CEP63 with 30 μl amylose resin)

  • Incubate with cell lysates containing GFP-tagged CEP152 (full-length, N-terminal, or C-terminal domains)

• Fluorescence microscopy:

  • Co-localization studies using anti-CEP63 and anti-CEP152 antibodies

  • Overexpression of CEP63 truncations (425-541 and 136-541) can deplete CEP152 from centrosomes

These approaches can be combined to comprehensively characterize the interaction domains and functional significance of the CEP63-CEP152 complex in centriole duplication.

What experimental approaches can effectively demonstrate CEP63's role in centriole duplication?

Several validated approaches can establish CEP63's function in centriole duplication:

• RNAi-mediated depletion:

  • siRNA treatment for 4 days effectively depletes CEP63

  • Quantify centrin foci in mitotic cells (normally 4 foci; 2 per centrosome)

  • CEP63 depletion leads to a significant increase in mitotic cells with fewer than 4 centrin foci

• Genetic models:

  • Gene-trap mouse models with insertion between exons 1 and 2 of the Cep63 gene

  • Mouse embryonic fibroblasts (MEFs) from these models show reduced centriole numbers

  • Homozygous gene-trap MEFs lack detectable CEP63 protein by immunofluorescence

• Centrosome reduplication assays:

  • Transfect cells with siRNAs, then add Aphidicolin (2 μg/ml)

  • Add second siRNA transfection after 24 hours

  • Collect cells after another 72 hours to assess centrosome numbers

These complementary approaches provide robust evidence for CEP63's essential role in ensuring reliable centriole duplication in dividing cells.

How do researchers optimize immunofluorescence protocols for CEP63 centrosomal localization?

Optimal visualization of CEP63 at centrosomes requires specific technical considerations:

• Image acquisition parameters:

  • Acquire Z-stacks at 0.2 μm intervals

  • Project by maximum intensity to a flat image

  • Apply deconvolution using "enhanced ratio (aggressive)" setting with medium noise filtering for 10 cycles

• Co-staining protocols for centrosomal markers:

  • For Centrin-2 and CEP63 co-staining: apply antibodies in specific order

    1. Anti-Centrin-2

    2. Goat anti-rabbit Alexa Fluor 594

    3. Anti-CEP63

    4. Goat anti-rabbit Alexa Fluor 488

• Equipment specifications:

  • Use high-resolution objectives (100×/1.4 oil recommended)

  • Recommended microscopy setup: Delta Vision RT inverted fluorescence microscope with Softworx software

  • Camera: COOLSNAPHQ/ICX285 CCD camera

Following these optimized protocols ensures specific detection of CEP63 at centrosomes while minimizing background interference and cross-reactivity issues.

What are the critical considerations when interpreting CEP63 knockdown phenotypes?

When analyzing CEP63 depletion experiments, researchers should consider:

• Phenotype specificity:

  • Similar phenotypes observed with CEP152 depletion suggest functional relationship

  • Analyze centriole numbers specifically in mitotic cells to control for cell cycle variations

• Verification methods:

  • Confirm knockdown efficiency by both immunofluorescence and Western blotting

  • For Western blot verification, CEP63 typically appears between 57-75 kDa

  • For immunofluorescence, compare centrosomal staining intensity to control cells

• Experimental timing:

  • RNAi effects are typically assessed after 4 days of treatment

  • For gene-trap models, consider potential developmental compensation mechanisms

• Quantification approach:

  • Count centrin foci in mitotic cells (normally 4; fewer indicates duplication defects)

  • Score multiple cells (n>60) across three independent experiments for statistical reliability

These considerations ensure accurate interpretation of CEP63 depletion phenotypes and their relevance to centriole duplication mechanisms.

What strategies can address non-specific binding in Western blots using CEP63 antibodies?

When encountering non-specific binding in Western blots, researchers can implement these troubleshooting strategies:

• Optimize blocking conditions:

  • Use 5% non-fat dry milk or BSA in TBST

  • Extend blocking time to 1-2 hours at room temperature

• Adjust antibody dilution:

  • For 16268-1-AP: Test dilutions at the higher end of the recommended range (closer to 1:2000)

  • For 66996-1-Ig: Start with 1:3000-1:6000 dilutions

• Washing optimization:

  • Increase wash duration and number of washes (5-6 washes, 5-10 minutes each)

  • Use fresh TBST buffer for each wash

• Sample preparation modifications:

  • Ensure complete protease inhibition during lysis

  • Consider centrifugation at higher speeds to remove debris

• Band identification:

  • CEP63 expected at 57-75 kDa despite calculated 81 kDa size

  • Multiple isoforms may appear as distinct bands

These adjustments can significantly reduce non-specific binding while maintaining strong specific CEP63 signal.

How can researchers differentiate between CEP63 isoforms in experimental systems?

To distinguish between the four CEP63 isoforms (56 kDa, 58 kDa, 63 kDa, and 81 kDa), researchers can employ:

• High-resolution gel electrophoresis:

  • Use 8% acrylamide gels for optimal separation in the 50-85 kDa range

  • Run gels at lower voltage for extended periods to maximize band separation

• Molecular weight verification:

  • Compare observed bands to recombinant CEP63 isoform standards

  • Use CEP63 truncation constructs as reference markers

• Western blot analysis:

  • Compare polyclonal (16268-1-AP) and monoclonal (66996-1-Ig) antibody staining patterns

  • Polyclonal antibody recognizes all four isoforms while monoclonal may show preference

• Cellular context:

  • Different cell types may express varying isoform profiles

  • Compare expression patterns across validated cell lines (HEK-293, HeLa, MCF-7)

These approaches enable researchers to characterize the specific CEP63 isoform expression patterns relevant to their experimental system.

What controls should be included when validating CEP63 antibody specificity?

Robust validation of CEP63 antibodies requires implementation of these essential controls:

• Negative controls:

  • CEP63 knockdown/knockout cells (RNAi or gene-trap)

  • Primary antibody omission controls

  • Isotype-matched irrelevant antibodies

• Positive controls:

  • Cell lines with validated expression (HEK-293, HeLa)

  • Tissues with confirmed expression (mouse liver, mouse thymus)

• Recombinant protein controls:

  • Tagged CEP63 constructs (YFP-Cep63, GFP-Cep63, Flag-Cep63)

  • CEP63 truncation constructs for domain specificity

• Technical validation:

  • Peptide competition assays

  • Comparison of staining patterns between different CEP63 antibodies

  • Colocalization with established centrosomal markers (γ-tubulin, Centrin-2)

These comprehensive controls establish antibody specificity and ensure experimental reproducibility across different applications.

What are the optimal conditions for immunoprecipitation of CEP63 complexes?

For successful isolation of CEP63 protein complexes, researchers should optimize:

• Antibody and lysate parameters:

  • Use 0.5-4.0 μg antibody per 1.0-3.0 mg total protein lysate

  • Incubate overnight at 4°C with gentle rotation

• Buffer composition for native complexes:

  • For MBP pull-down assays: column buffer (20 mM Tris HCl pH7.5, 200 nM NaCl, 1 mM EDTA, 1 mM DTT)

  • Include Complete protease inhibitors (Roche)

• Experimental strategies for tagged proteins:

  • For Flag-tagged CEP63: Anti-Flag resin works efficiently

  • For MBP-tagged CEP63: Use 2 μg protein with 30 μl amylose resin

• Washing conditions:

  • For native IP: PBS washes (3-5 times)

  • For pull-down assays: Column buffer washes followed by PBS

• Elution methods:

  • For analytical purposes: Boil in Laemmli buffer

  • For functional studies: Elute with appropriate competing agents (e.g., Flag peptide)

These optimized conditions ensure efficient isolation of CEP63 and its interacting partners while minimizing non-specific interactions.