ECP63 Antibody

What is CEP63 and what cellular functions does it perform?

CEP63 is a 63 kDa centrosomal protein that serves multiple critical cellular functions. It is required for normal spindle assembly during cell division and maintains proper centrosome numbers through centrosomal recruitment of CEP152. Additionally, CEP63 recruits CDK1 to centrosomes and plays a significant role in DNA damage response mechanisms .

The protein exists in four isoforms (56 kDa, 58 kDa, 63 kDa, and 81 kDa), with a calculated molecular weight of 81 kDa (703 amino acids) but an observed molecular weight typically between 57-75 kDa in experimental settings . Following DNA damage events such as double-strand breaks, CEP63 is removed from centrosomes, which leads to inactivation of spindle assembly and delay in mitotic progression . Recent research has also identified that CEP63 promotes stabilization of FXR1 protein by inhibiting its ubiquitination .

What applications can CEP63 antibodies be used for?

CEP63 antibodies have been validated for multiple research applications, with specific performance characteristics for each application. The primary applications include:

It's important to note that application-specific optimization is necessary as performance can vary based on sample type, fixation method, and experimental conditions .

How should I validate the specificity of a CEP63 antibody?

Validating antibody specificity is critical for ensuring reliable experimental results. For CEP63 antibodies, implement a comprehensive validation approach including:

The implementation of these validation strategies is critical, as inadequately characterized antibodies have led to significant reproducibility issues in research. A concerning example from the literature demonstrates how an unvalidated antibody (unrelated to CEP63) was used in 15 publications, generating over 3,000 citations despite not recognizing its putative target .

What are the best approaches for optimizing CEP63 antibody performance in challenging experimental conditions?

Optimizing CEP63 antibody performance in challenging experimental conditions requires a systematic approach:

• Antigen retrieval optimization for IHC-P: For formalin-fixed paraffin-embedded sections, heat-mediated antigen retrieval in citrate buffer (pH 6.0) for 20 minutes has proven effective for exposing CEP63 epitopes . Compare multiple antigen retrieval methods (citrate, EDTA, enzymatic) if initial results are suboptimal.

• Buffer and blocking optimization for Western blot:

  • Test multiple blocking agents (5% non-fat milk, 5% BSA, commercial blockers)

  • Evaluate different wash buffer compositions (varying Tween-20 concentrations)

  • Titrate primary antibody concentrations between 1:500-1:2000

  • Vary incubation times and temperatures (overnight at 4°C vs. 1-2 hours at room temperature)

• Signal amplification strategies: For low-abundance detection, implement tyramide signal amplification (TSA) or polymer-based detection systems, which can increase sensitivity 10-100 fold over conventional detection methods.

• Sample preparation considerations: For centrosomal proteins like CEP63, incorporating a pre-extraction step with detergents before fixation can remove cytoplasmic proteins and enhance centrosome visualization in immunofluorescence applications.

• Empirical testing across applications: Performance of CEP63 antibodies varies by application. For example, the GTX634482 antibody has been shown to work optimally for immunoblot and immunohistochemistry but lacks specificity in immunofluorescence and fails completely in immunoprecipitation applications .

How do post-translational modifications of CEP63 affect antibody detection?

Post-translational modifications (PTMs) can significantly affect CEP63 antibody binding and detection:

• Phosphorylation-dependent epitope masking: CEP63 undergoes phosphorylation during DNA damage response, which may alter antibody recognition. For comprehensive detection, use phosphorylation-independent antibodies or multiple antibodies targeting different regions.

• Conformational changes: PTMs can induce conformational changes that mask or expose different epitopes. Some antibodies recognize linear epitopes while others detect conformational epitopes, necessitating empirical testing for each application .

• Technical considerations:

  • Add phosphatase inhibitors (sodium orthovanadate, sodium fluoride) to lysis buffers when studying phosphorylated CEP63

  • Consider lambda phosphatase treatment as a control to verify phosphorylation-dependent detection

  • For ubiquitination studies, include deubiquitinating enzyme inhibitors and perform IP under denaturing conditions

• Application-specific considerations: For immunofluorescence applications, pre-extractable versus non-extractable fractions of CEP63 may represent different PTM populations, necessitating optimization of fixation and extraction protocols.

What strategies should be employed for simultaneous detection of CEP63 and interacting proteins?

Effective multi-protein detection requires careful experimental design:

• Co-immunoprecipitation optimization:

  • Use mild lysis conditions (150mM NaCl, 0.5% NP-40) to preserve protein-protein interactions

  • For CEP63-CEP152 interactions, optimize antibody amounts (0.5-4.0 μg per 1.0-3.0 mg total protein)

  • Include appropriate controls (IgG control, reverse IP)

• Multi-color immunofluorescence strategies:

  • Select primary antibodies from different host species (rabbit anti-CEP63 combined with mouse anti-CEP152)

  • For same-species antibodies, use direct conjugation or sequential detection with Fab fragments

  • Implement spectral unmixing for overlapping fluorophores

• Proximity ligation assay (PLA):

  • Consider PLA for detecting CEP63 interactions with CDK1, CEP152, or other centrosomal proteins

  • This technique provides single-molecule resolution of protein interactions (<40nm proximity)

  • Requires specific primary antibodies and species-appropriate PLA probes

• Controls for co-localization studies:

  • Include biological controls (siRNA knockdown)

  • Consider image analysis controls (randomized image rotation to assess statistical significance of co-localization)

  • Implement quantitative co-localization metrics (Pearson's correlation, Manders' coefficients)

What are the considerations for using CEP63 antibodies across different species models?

When working with CEP63 antibodies across different model organisms, several factors must be considered:

Key considerations for cross-species applications:

• Epitope conservation analysis: Before using a CEP63 antibody in a new species, analyze sequence homology in the epitope region. Higher homology increases likelihood of cross-reactivity.

• Validation requirements: Always validate antibodies in the specific species of interest using positive and negative controls:

  • Positive control: tissue known to express CEP63 (liver, thymus for mouse)

  • Negative control: CEP63 knockdown or knockout samples

  • Method-specific controls: preabsorption with immunizing peptide

• Species-specific protocol adjustments:

  • Adjust fixation protocols based on species (4% PFA for 10 minutes for human cells vs. 15 minutes for mouse tissues)

  • Modify blocking conditions (species-matched normal serum)

  • Optimize primary antibody concentrations and incubation times

• Western blot considerations: Expected molecular weight may vary slightly between species due to sequence differences and post-translational modifications.

How can I troubleshoot non-specific binding with CEP63 antibodies?

Non-specific binding is a common challenge when working with antibodies. For CEP63 antibodies, implement these troubleshooting strategies:

• Western blot non-specific bands:

  • Increase blocking stringency (5% BSA or commercial blockers instead of milk)

  • Titrate antibody concentration (test dilutions between 1:500-1:2000)

  • Increase wash duration and number of wash steps

  • Consider using different secondary antibody

  • Perform peptide competition assay to identify specific bands

• Immunofluorescence background:

  • Optimize fixation protocols (test 4% PFA vs. methanol fixation)

  • Include detergent pre-extraction step to remove cytoplasmic proteins

  • Increase blocking time and concentration (add 5% normal serum from secondary antibody host)

  • Test different mounting media to reduce background

  • Consider autofluorescence quenching steps

• Immunoprecipitation non-specific pull-down:

  • Pre-clear lysates with protein A/G beads

  • Use IgG controls from the same species as the antibody

  • Optimize antibody amount (0.5-4.0 μg range recommended)

  • Adjust wash buffer stringency (increase salt concentration)

• Cross-reactivity assessment: Different antibodies show variable specificity across applications. As noted in search results, some CEP63 antibodies work well in certain applications but poorly in others .

What are the critical quality control parameters for CEP63 antibody experiments?

Implementing rigorous quality control is essential for reliable CEP63 antibody experiments:

• Antibody validation controls:

  • Positive control: Tissues/cells known to express CEP63 (HEK-293, HeLa, mouse liver, mouse thymus)

  • Negative control: CEP63 knockdown/knockout samples

  • Loading controls: Appropriate housekeeping proteins for Western blot

  • Immunofluorescence controls: Secondary-only controls, IgG controls, competing peptide controls

• Batch-to-batch consistency testing:

  • Document lot numbers and test new lots against previous validated lots

  • Maintain reference samples for comparison

  • Consider aliquoting antibodies to avoid freeze-thaw cycles

• Storage and handling verification:

  • Store at -20°C for long-term storage

  • Avoid repeated freeze-thaw cycles

  • Follow manufacturer's recommendations for stability (generally stable for one year after shipment)

  • For 20μl sizes, note that they contain 0.1% BSA as a stabilizer

• Method-specific quality controls:

  • Western blot: Molecular weight markers, expected size range (57-75 kDa)

  • Immunofluorescence: Co-staining with established centrosomal markers

  • Immunoprecipitation: Input, flow-through, and IgG control samples

How should CEP63 antibody data be quantified and analyzed for reproducible results?

Proper quantification and analysis of CEP63 antibody data is crucial for reproducible research:

• Western blot quantification:

  • Use digital image acquisition with linear dynamic range

  • Implement normalization to appropriate loading controls

  • Apply lane background subtraction

  • Present data as relative abundance rather than absolute values

  • Test multiple exposure times to ensure signal is in linear range

• Immunofluorescence quantification:

  • For centrosomal CEP63, measure fluorescence intensity within defined regions

  • Use automated detection algorithms for unbiased quantification

  • Consider 3D analysis for complete centrosome volume assessment

  • Establish intensity thresholds consistently across experimental conditions

  • Analyze sufficient cell numbers for statistical power (minimum 50-100 cells per condition)

• Reproducibility considerations:

  • Report RRID (Research Resource Identifier) for antibodies (e.g., RRID:AB_2077079 for CEP63)

  • Document detailed experimental protocols

  • Report antibody dilutions, incubation times and temperatures

  • Include biological and technical replicates (minimum n=3)

  • Apply appropriate statistical tests based on data distribution

• Standard curve approaches:

  • For quantitative applications, develop standard curves using recombinant CEP63 protein

  • Report data with appropriate error bars and statistical significance

  • Consider blinding analysis to reduce unconscious bias

How can CEP63 antibodies be used to study centrosome duplication in cancer research?

CEP63 plays a crucial role in centrosome duplication, making it an important target in cancer research where centrosome abnormalities are common:

• Quantitative assessment of centrosome amplification:

  • Use CEP63 antibodies (1:200-1:800 dilution for IF) in multi-color immunofluorescence with γ-tubulin or centrin

  • Implement high-content imaging to score centrosome number per cell across large populations

  • Compare centrosome numbers in normal versus cancer cell lines

  • Correlate with cell cycle markers to distinguish duplication defects from cytokinesis failures

• DNA damage response studies:

  • Monitor CEP63 localization following DNA damage induced by radiation or genotoxic agents

  • As shown in research, CEP63 is removed from centrosomes following DNA damage, leading to spindle assembly inactivation

  • Combine with γH2AX staining to correlate with double-strand break formation

  • Use time-lapse imaging to track dynamics of CEP63 dissociation from centrosomes

• Functional analysis in cancer models:

  • Correlate CEP63 levels with cancer stage and grade

  • Analyze CEP63 immunohistochemistry in tissue microarrays

  • Integrate with genomic data on CEP63 mutations or amplifications

  • Develop tissue processing protocols that preserve centrosomal architecture

• Therapeutic target assessment:

  • Use CEP63 antibodies to evaluate effects of centrosome-targeting drugs

  • Monitor changes in CEP63-CEP152 interactions following drug treatment

  • Implement proximity ligation assays to quantify protein interaction changes

What approaches can be used to study CEP63's role in the DNA damage response pathway?

CEP63's involvement in DNA damage response pathways can be investigated using these advanced approaches:

• Damage-induced translocation studies:

  • Perform time-course experiments following DNA damage induction

  • Use immunofluorescence (1:200-1:800 dilution) to track CEP63 localization changes

  • Combine with staining for ATM/ATR pathway components

  • Apply super-resolution microscopy for detailed localization analysis

  • Quantify centrosomal versus non-centrosomal CEP63 fractions

• Protein modification analysis:

  • Use immunoprecipitation (0.5-4.0 μg antibody) followed by Western blot analysis for post-translational modifications

  • Probe for phosphorylation events using phospho-specific antibodies

  • Investigate ubiquitination status following DNA damage

  • Perform mass spectrometry analysis of immunoprecipitated CEP63 to identify novel modifications

• Functional rescue experiments:

  • Deplete endogenous CEP63 using siRNA

  • Re-express siRNA-resistant wild-type or mutant CEP63

  • Assess rescue of centrosome duplication and DNA damage response phenotypes

  • Use antibody detection to confirm appropriate expression levels

• Cell cycle synchronization approaches:

  • Synchronize cells at different cell cycle phases

  • Analyze CEP63 levels and localization across the cell cycle

  • Combine with DNA damage at specific cell cycle stages

  • Quantify nuclear versus centrosomal fractions of CEP63

How can CEP63 antibodies contribute to understanding developmental disorders associated with centrosomal dysfunction?

CEP63 mutations have been implicated in microcephaly and other developmental disorders linked to centrosomal dysfunction:

• Patient-derived cell analysis:

  • Compare CEP63 levels and localization in patient-derived cells versus controls

  • Assess centrosome structure and number using CEP63 antibodies in combination with other centrosomal markers

  • Quantify centrosome duplication efficiency across multiple cell cycles

  • Analyze mitotic spindle organization and chromosome segregation

• Model system approaches:

  • Use CEP63 antibodies in developmental studies of model organisms

  • Perform immunohistochemistry on developing brain tissues

  • Correlate CEP63 expression with neuronal differentiation markers

  • Apply validated antibodies across species (human, mouse, rat)

• iPSC and organoid applications:

  • Generate iPSCs from patients with CEP63 mutations

  • Differentiate into neural progenitors and mature neurons

  • Track CEP63 expression and localization during differentiation

  • Apply immunofluorescence protocols optimized for 3D organoid structures

• Therapeutic strategy assessment:

  • Evaluate potential rescue strategies in patient-derived cells

  • Use CEP63 antibodies to monitor restoration of normal centrosome function

  • Combine with functional readouts of cell division and differentiation

  • Implement high-content screening approaches for therapeutic discovery

What emerging technologies are enhancing CEP63 antibody applications?

Several cutting-edge technologies are expanding the capabilities of CEP63 antibody applications:

• Super-resolution microscopy applications:

  • Implement STED, SIM, or STORM microscopy for nanoscale resolution of CEP63 localization

  • Resolve sub-centrosomal structures with 20-50nm precision

  • Combine with proximity labeling techniques for protein interaction mapping

  • Apply quantitative cluster analysis for molecular distribution patterns

• Single-cell proteomics integration:

  • Combine CEP63 antibody staining with mass cytometry (CyTOF)

  • Integrate with single-cell RNA-seq data for multi-omic analysis

  • Implement imaging mass cytometry for tissue-level analysis

  • Correlate CEP63 protein levels with transcriptional states

• Live-cell imaging adaptations:

  • Develop antibody-based biosensors for CEP63 dynamics

  • Apply nanobody technology for improved intracellular penetration

  • Implement optogenetic approaches combined with antibody detection

  • Use FRET-based sensors to monitor CEP63 interactions in real-time

• Antibody engineering advancements:

  • Harness recombinant antibody technologies for improved reproducibility

  • Apply phage display for epitope-specific antibody generation

  • Develop bivalent antibodies for enhanced avidity

  • Create antibody fragments with improved tissue penetration properties

How can researchers contribute to improved standardization of CEP63 antibody research?

Researchers can advance CEP63 antibody standardization through these approaches:

• Comprehensive validation reporting:

  • Document validation using multiple methods (genetic, biochemical, orthogonal)

  • Publish detailed validation protocols and results

  • Register antibodies with RRID identifiers (e.g., RRID:AB_2077079)

  • Contribute to public antibody validation repositories

• Methodological standardization:

  • Establish standard operating procedures for common applications

  • Report detailed methods including antibody concentration, incubation conditions, and buffers

  • Perform inter-laboratory validation studies

  • Develop application-specific positive and negative controls

• Open science practices:

  • Share raw image data in public repositories

  • Provide detailed protocols on platforms like protocols.io

  • Report negative results to counter publication bias

  • Participate in community-driven antibody validation initiatives

• Advanced validation approaches:

  • Implement multiple antibody validation criteria as recommended in the literature

  • Prioritize genetic validation strategies using CRISPR/Cas9 knockout controls

  • Perform immunoprecipitation-mass spectrometry validation

  • Contribute validation data to antibody databases and repositories