CEP70 Antibody

What is CEP70 and what are its primary cellular functions?

CEP70 (Centrosomal protein of 70 kDa) is a component of the centrosome that plays critical roles in microtubule organization. Specifically, CEP70:

• Participates in organizing both preexisting and nascent microtubules during interphase

• Is required for the organization and orientation of the mitotic spindle during mitosis

• Interacts within a complex network of proteins including CEP135 and γ-tubulin

• Facilitates the stabilization of microtubules and supports spindle microtubule nucleation

CEP70 localizes to the centrosome throughout the cell cycle, as demonstrated by immunofluorescence studies showing colocalization with centrosomal markers like γ-tubulin in both interphase and mitotic cells .

What is the molecular structure of CEP70 and how does it influence its function?

CEP70 is a 597 amino acid protein with a calculated molecular weight of approximately 70 kDa. The protein contains multiple coiled-coil domains that are critical for its function and localization:

• The peptide fragments containing the coiled-coil domains are essential for CEP70's interaction with γ-tubulin

• These domains are also crucial for proper localization of CEP70 to the centrosome

• Truncation studies have shown that amino acid sequences 1-597 (full-length), 1-326, and 75-326 of CEP70 interact with γ-tubulin, while sequences 75-179, 254-326, 1-179, and 327-597 do not interact

This structural organization suggests that the coiled-coil domains create interaction surfaces that mediate both protein-protein interactions and centrosomal targeting.

What criteria should I use when selecting a CEP70 antibody for my specific application?

When selecting a CEP70 antibody, consider the following research-focused criteria:

• Application compatibility: Determine if the antibody has been validated for your intended application (WB, IHC, ICC/IF, IP)

• Species reactivity: Confirm reactivity with your experimental model organism (human, mouse, etc.)

• Epitope location: Some antibodies target specific regions (e.g., N-terminal) which may affect detection based on protein conformation or interactions

• Validation data: Review available immunoblotting, IHC, and IF data to assess specificity

• Immunogen information: Check whether the antibody was raised against a recombinant fragment or synthetic peptide, which may influence epitope recognition

For instance, antibody ab237801 is suitable for WB, IHC-P, and ICC/IF applications and reacts with human and mouse samples, with the immunogen corresponding to a recombinant fragment within human CEP70 aa 50-300 .

How can I validate the specificity of my CEP70 antibody before conducting critical experiments?

A rigorous validation approach for CEP70 antibodies should include:

• Positive and negative controls:

  • Use cell lines known to express CEP70 (e.g., HeLa, MCF7, PC-3) as positive controls

  • Include non-expressing or knockdown samples as negative controls

• Multiple detection methods:

  • Western blot: Confirm single band of expected size (~70 kDa, though some isoforms exist at 67, 65, and 25 kDa)

  • Immunofluorescence: Verify centrosomal localization with colocalization studies using established centrosomal markers like γ-tubulin or pericentrin

• siRNA knockdown:

  • Transfect cells with CEP70-specific siRNAs and confirm reduced signal by Western blot and immunofluorescence

  • Use non-targeting siRNA as control

• Overexpression studies:

  • Express tagged CEP70 (e.g., GFP-Cep70) and confirm antibody recognition

Following these validation steps will ensure antibody specificity before proceeding with critical experiments.

What are the optimal conditions for using CEP70 antibodies in immunofluorescence microscopy?

Based on established protocols from the literature, optimal conditions for CEP70 immunofluorescence include:

• Fixation method:

  • Methanol fixation at -20°C is recommended for optimal centrosomal staining

  • This preserves centrosome structure while providing good antigen accessibility

• Blocking conditions:

  • 2% bovine serum albumin (BSA) in PBS to reduce background

• Antibody dilutions:

  • Primary antibody: Generally 1:50-1:500 (specific recommendations: ab237801 at 1:100, ab227456 at 1:500)

  • Use fluorescein- or rhodamine-conjugated secondary antibodies

• Counterstaining:

  • DAPI for nuclear visualization

  • Co-staining with γ-tubulin or pericentrin to confirm centrosomal localization

• Microscopy technique:

  • Standard fluorescence microscopy for general localization

  • Confocal microscopy for detailed spindle morphology analysis

For accurate intensity measurements of CEP70 at the centrosome, utilize the two-square measurement method: center two computer-generated squares on each centrosome, with the inner square containing the CEP70 signal plus background, and the region between the squares representing background only. Subtract the background to obtain the true CEP70 signal intensity .

What protocol modifications are needed for optimal Western blot detection of CEP70?

For successful Western blot detection of CEP70, consider these technical modifications:

• Sample preparation:

  • Use whole cell lysates from tissues or cultured cells (human placenta, heart, kidney, PC-3, MCF7)

  • Include protease inhibitors to prevent degradation

• Gel percentage:

  • Use 7.5% SDS-PAGE for optimal separation of the 70 kDa protein

• Transfer conditions:

  • Standard transfer protocols for proteins in this size range

• Antibody concentrations:

  • Primary antibody dilutions: 1:1000-1:4000 (specific recommendations: ab237801 at 1:1000, 16280-1-AP at 1:1000-1:4000)

  • Secondary antibody: Anti-rabbit IgG at approximately 1:50000 dilution

• Expected band sizes:

  • Main band at approximately 70 kDa (predicted size)

  • Be aware that CEP70 can exist in multiple isoforms with molecular weights of 70, 67, 65, and 25 kDa

• Positive controls:

  • Human placenta lysate

  • PC-3 (human prostate adenocarcinoma) whole cell lysate

  • Mouse heart and kidney lysates have shown reliable detection

These modifications will enhance detection sensitivity and specificity when working with CEP70.

How can I effectively use CEP70 antibodies to study centrosome dynamics during the cell cycle?

To effectively study centrosome dynamics using CEP70 antibodies:

• Cell synchronization techniques:

  • Use nocodazole treatment for mitotic arrest

  • Thymidine block for S-phase synchronization

  • Serum starvation for G0/G1 enrichment

• Time-course experiments:

  • Fix cells at different cell cycle phases

  • Use CEP70 antibodies (1:100 dilution) with co-staining for cell cycle markers

  • Track CEP70 intensity and localization changes through the cell cycle

• Live-cell imaging:

  • Combine with fluorescently-tagged CEP70 constructs

  • Use stable cell lines expressing GFP-CEP70 at near-endogenous levels

• Quantitative analysis:

  • Measure CEP70 intensity at the centrosome using the two-square method described previously

  • Track changes in intensity across cell cycle stages

  • Correlate with functional assays of microtubule organization

• Functional perturbation:

  • Use siRNA knockdown of CEP70 to observe effects on centrosome structure and function

  • Compare phenotypes with those observed in γ-tubulin or pericentrin depletion experiments

This integrated approach will provide insights into how CEP70 contributes to dynamic centrosome functions throughout the cell cycle.

What experimental strategies can help resolve contradictory results when studying CEP70 expression in cancer tissues?

When facing contradictory results regarding CEP70 expression in cancer:

• Validation across multiple detection methods:

  • Compare results from immunohistochemistry, Western blot, and qRT-PCR

  • Use at least two different validated antibodies targeting different epitopes of CEP70

  • Include proper controls for each technique

• Comprehensive tissue sampling:

  • Analyze multiple regions within tumors to account for heterogeneity

  • Include paired normal adjacent tissue samples

  • Correlate with clinicopathological parameters (histological grade, pTNM stage, LN metastasis)

• Standardized scoring systems:

  • Implement quantitative intensity measurement for immunohistochemistry

  • Use histological scoring systems (H-score or Allred)

  • Blind analysis by multiple pathologists

• Cell line validation:

  • Compare expression across multiple cancer cell lines (e.g., AsPC1, PANC1, CFPAC1, BxPC3 for pancreatic cancer)

  • Validate with functional assays (proliferation, migration)

• Genetic verification:

  • Assess gene copy number to rule out amplification effects

  • Investigate epigenetic modifications (promoter methylation, histone modifications)

A study on pancreatic cancer demonstrated that while CEP70 mRNA expression was elevated, gene copy number remained unchanged, pointing to potential epigenetic mechanisms of overexpression .

How can CEP70 antibodies be used to investigate the role of this protein in cancer progression?

To investigate CEP70's role in cancer progression:

• Expression correlation studies:

  • Use immunohistochemistry with CEP70 antibodies (1:100 dilution) on tissue microarrays

  • Compare expression between normal and cancerous tissues

  • Correlate with clinical parameters and survival data

  Parameter	Correlation with CEP70 Expression
  Histological grade	Significant correlation observed
  pTNM stage	Significant correlation observed
  Lymph node metastasis	Significant correlation observed
  CA19-9 levels	Correlation observed

  For instance, in pancreatic cancer, only 7.4% of normal pancreatic tissues showed high CEP70 expression, while 77.6% of cancer tissues demonstrated high expression .

• Functional studies in cancer cell models:

  • Knockdown CEP70 using specific siRNAs in cancer cell lines

  • Assess effects on proliferation using sulforhodamine B staining

  • Evaluate impact on cell cycle progression through BrdU incorporation

  • Analyze migration and invasion capabilities

• Mechanistic investigations:

  • Analyze centrosome amplification and spindle abnormalities using CEP70 antibodies

  • Examine effects on chromosomal stability

  • Investigate downstream signaling pathways affected by CEP70 modulation

• Therapeutic potential assessment:

  • Evaluate CEP70 as a potential biomarker for specific cancer types

  • Test the efficacy of targeting CEP70 in combination with standard therapies

These approaches can provide comprehensive insights into CEP70's role in cancer biology and its potential as a therapeutic target .

What are the most common technical issues when using CEP70 antibodies and how can they be resolved?

Common technical issues with CEP70 antibodies and their solutions include:

• Low or no signal in Western blot:

  • Increase antibody concentration (try 1:500 instead of 1:1000)

  • Extend primary antibody incubation time (overnight at 4°C)

  • Use enhanced chemiluminescence detection systems

  • Verify protein loading and transfer efficiency

  • Consider different extraction buffers to improve solubilization of centrosomal proteins

  • Be aware that CEP70 expression varies between cell types; 293T cells may show low abundance

• High background in immunofluorescence:

  • Increase blocking time and concentration (try 5% BSA)

  • Optimize antibody dilution (start with 1:100 and adjust)

  • Include additional washing steps

  • Use highly cross-adsorbed secondary antibodies

  • Consider alternative fixation methods if methanol fixation gives poor results

• Multiple bands in Western blot:

  • Be aware that CEP70 exists in multiple isoforms (70, 67, 65, and 25 kDa)

  • Verify if bands disappear with siRNA treatment

  • Use positive control lysates with known CEP70 expression (human placenta, PC-3 cells)

• Non-specific centrosomal staining:

  • Co-stain with established centrosomal markers (γ-tubulin, pericentrin)

  • Verify specificity through siRNA knockdown

  • Use super-resolution microscopy for better localization precision

These troubleshooting approaches will help resolve common technical challenges when working with CEP70 antibodies.

How should researchers interpret and validate unusual subcellular localization patterns of CEP70?

When encountering unusual CEP70 localization patterns:

• Verification strategies:

  • Confirm with multiple antibodies targeting different epitopes

  • Use fluorescently-tagged CEP70 constructs to validate localization

  • Apply super-resolution microscopy techniques for precise localization

  • Conduct fractionation studies to biochemically confirm localization

• Context-dependent interpretation:

  • Consider cell cycle stage (CEP70 normally localizes to centrosomes throughout the cell cycle)

  • Assess if the cell is undergoing normal or abnormal mitosis

  • Evaluate potential post-translational modifications affecting localization

  • Determine if observed localization changes under experimental conditions

• Co-localization studies:

  • Perform detailed co-localization with multiple centrosomal markers

  • Include markers for various subcellular compartments to identify non-centrosomal localization

  • Calculate co-localization coefficients for quantitative assessment

• Functional validation:

  • Use domain deletion constructs to identify regions responsible for unusual localization

  • Examine if protein-protein interactions are altered in the unusual localization context

  • Test if the unusual localization correlates with functional changes in microtubule organization

These approaches provide a framework for validating and interpreting unexpected CEP70 localization patterns in a scientifically rigorous manner .

How can CEP70 antibodies be utilized in studying centrosome amplification in cancer cells?

For studying centrosome amplification in cancer:

• Quantitative immunofluorescence approach:

  • Use CEP70 antibodies (1:100 dilution) with γ-tubulin co-staining

  • Count centrosome number per cell (>2 centrosomes indicates amplification)

  • Calculate percentage of cells with amplified centrosomes

  • Compare normal versus cancer cell lines or tissues

• High-content imaging and analysis:

  • Develop automated image acquisition and analysis workflows

  • Quantify not only centrosome number but also size, intensity, and clustering

  • Correlate with cell cycle markers and chromosomal aberrations

• 3D imaging techniques:

  • Apply confocal z-stack imaging to visualize the full centrosome structure

  • Use 3D reconstruction to accurately assess centrosome clustering in cancer cells

• Functional consequences assessment:

  • Combine with live-cell imaging to track mitotic progression and errors

  • Monitor chromosome segregation defects using DAPI staining

  • Correlate centrosome amplification with aneuploidy and genomic instability

• Therapeutic screening platform:

  • Utilize CEP70 antibodies to evaluate centrosome-targeting drugs

  • Assess centrosome clustering inhibitors effectiveness

  • Screen for compounds that selectively target cells with centrosome amplification

This methodology can provide valuable insights into the role of centrosome amplification in cancer development and progression .

What are the latest methodological advances in using CEP70 antibodies for studying microtubule organization?

Recent methodological advances include:

• Super-resolution microscopy techniques:

  • STED (Stimulated Emission Depletion) microscopy for nanoscale resolution of centrosome structure

  • SIM (Structured Illumination Microscopy) for improved visualization of microtubule-centrosome interactions

  • Single-molecule localization microscopy for precise mapping of CEP70 within the centrosome

• Live-cell imaging approaches:

  • Combine with photoactivatable or photoswitchable fluorescent protein-tagged CEP70

  • Track dynamic changes in CEP70 localization during microtubule reorganization

  • Measure recruitment kinetics of CEP70 during centrosome maturation

• Proximity labeling techniques:

  • BioID or APEX2 fusion with CEP70 to identify proximal interacting proteins

  • Compare interactomes between normal and pathological conditions

  • Map the spatial organization of the centrosomal protein network

• Correlative light and electron microscopy (CLEM):

  • Combine immunofluorescence using CEP70 antibodies with electron microscopy

  • Precisely localize CEP70 within ultrastructural context of the centrosome

  • Map relationships between CEP70, centrioles, and pericentriolar material

These advanced methodologies enable researchers to gain unprecedented insights into the spatial and temporal dynamics of CEP70 in microtubule organization .

How should researchers design experiments to investigate the relationship between CEP70 expression and cancer prognosis?

To investigate CEP70 expression and cancer prognosis:

This comprehensive approach was utilized in pancreatic cancer research, where CEP70 expression showed significant correlation with histological grade, pTNM stage, lymph node metastasis, and CA19-9 levels .

What methodological considerations are important when investigating CEP70's potential as a therapeutic target?

When investigating CEP70 as a therapeutic target:

• Target validation strategy:

  • Confirm overexpression in disease tissues compared to normal using antibody-based detection

  • Validate effects of genetic knockdown (siRNA, CRISPR) on cancer cell phenotypes

  • Conduct rescue experiments to confirm specificity of observed effects

  • Assess effects in multiple cell lines to ensure generalizability

• Mechanism of action studies:

  • Identify downstream pathways affected by CEP70 modulation

  • Characterize effects on centrosome function, microtubule organization, and mitotic progression

  • Evaluate impacts on chromosomal stability and genomic integrity

  • Use CEP70 antibodies to monitor centrosome changes after intervention

• Therapeutic approach selection:

  • Consider small molecule inhibitors targeting protein-protein interactions

  • Evaluate antisense oligonucleotides or siRNA-based therapeutics

  • Assess antibody-drug conjugates targeting CEP70-expressing cells

  • Develop combination approaches with standard chemotherapeutics

• Toxicity and specificity assessment:

  • Test effects on normal cells with lower CEP70 expression

  • Evaluate potential off-target effects

  • Conduct detailed analysis of centrosome and mitotic spindle integrity

• Translational considerations:

  • Develop companion diagnostics using validated CEP70 antibodies

  • Identify patient populations most likely to benefit from CEP70-targeting

  • Design appropriate clinical endpoints based on mechanism of action

These methodological considerations provide a framework for investigating CEP70 as a potential therapeutic target in cancer, as suggested by findings in pancreatic cancer research .