CKAP5 Antibody

What is CKAP5 and why is it important in cellular research?

CKAP5 (Cytoskeleton Associated Protein 5) is a microtubule-associated protein that plays a crucial role in mitotic spindle assembly by affecting tubulin functions and facilitating centrosomal fragmentation . The canonical human protein consists of 2032 amino acid residues with a molecular mass of approximately 225.5 kDa . As a member of the TOG/XMAP215 protein family, CKAP5 binds to the plus end of microtubules and regulates microtubule dynamics and organization . It is particularly notable for its expression in hepatomas and colonic tumors, making it a significant target in cancer research . The protein has several synonyms in the literature, including MSPS, TOG, TOGp, ch-TOG, colonic and hepatic tumor over-expressed gene protein, and CHTOG .

What are the most common applications for CKAP5 antibodies in research?

CKAP5 antibodies are primarily utilized in several key laboratory techniques:

• Western Blot (WB): The most widely used application for detecting and quantifying CKAP5 protein expression in cell or tissue lysates .

• Immunofluorescence (IF)/Immunocytochemistry (ICC): Frequently used to visualize the subcellular localization of CKAP5, particularly its association with microtubules and centrosomes during various cell cycle stages .

• Immunoprecipitation (IP): Employed to isolate CKAP5 and its binding partners to study protein-protein interactions .

• Flow Cytometry (FCM): Used to detect and quantify CKAP5 expression at the single-cell level .

• Immunohistochemistry (IHC): Applied to visualize CKAP5 expression patterns in tissue sections, particularly in tumor samples .

What should researchers consider when selecting a CKAP5 antibody?

When selecting a CKAP5 antibody for research, consider:

• Antibody specificity: Ensure the antibody specifically recognizes CKAP5 without cross-reactivity to other proteins, particularly other TOG family members.

• Species reactivity: Verify that the antibody recognizes CKAP5 in your experimental species (human, mouse, rat, etc.) .

• Application validation: Confirm the antibody has been validated for your specific application (WB, IF, IHC, etc.) .

• Isoform recognition: Since CKAP5 has up to three different isoforms, determine which isoform(s) the antibody recognizes .

• Clonality: Consider whether a monoclonal or polyclonal antibody is more appropriate for your specific research question.

• Citations: Review literature citations to evaluate the antibody's performance in contexts similar to your planned experiments .

What controls should be included when using CKAP5 antibodies?

For rigorous experimental design with CKAP5 antibodies, include:

• Positive control: Lysates or samples from tissues/cells known to express CKAP5 (e.g., hepatomas, colonic tumors) .

• Negative control: Samples where CKAP5 is known to be absent or samples with CKAP5 knockdown/knockout .

• Primary antibody omission: To detect non-specific binding of secondary antibodies.

• Isotype control: An irrelevant antibody of the same isotype as the CKAP5 antibody.

• Blocking peptide: If available, use a specific CKAP5 peptide to demonstrate antibody specificity.

• siRNA treatment control: Cells treated with CKAP5-targeting siRNA to confirm antibody specificity .

How should CKAP5 antibodies be optimized for Western blot analysis?

For optimal Western blot results with CKAP5 antibodies:

• Protein extraction: Use buffers containing phosphatase and protease inhibitors to preserve CKAP5 integrity, as it is a large protein susceptible to degradation.

• Sample preparation: Heat samples at 70°C instead of 95°C to prevent aggregation of this large protein.

• Gel selection: Use low-percentage (6-8%) gels or gradient gels to properly resolve this 225.5 kDa protein .

• Transfer conditions: Employ wet transfer with extended duration (overnight at low voltage) to ensure complete transfer of this high molecular weight protein.

• Antibody dilution: Typically start with 1:1000 dilution and optimize based on signal-to-noise ratio.

• Blocking conditions: Use 5% BSA in TBST rather than milk, as milk can sometimes interfere with detection of certain epitopes.

• Detection method: Consider enhanced chemiluminescence with extended exposure times for optimal visualization.

What are the best approaches for visualizing CKAP5 in immunofluorescence experiments?

For successful immunofluorescence visualization of CKAP5:

• Fixation method: Use 4% paraformaldehyde for 15-20 minutes at room temperature to preserve microtubule structures.

• Permeabilization: Apply 0.1-0.2% Triton X-100 for adequate antibody access to cytoplasmic CKAP5.

• Blocking: Block with 5% normal serum from the species of the secondary antibody for 1 hour.

• Primary antibody incubation: Incubate with optimized CKAP5 antibody dilution (typically 1:100 to 1:500) overnight at 4°C.

• Co-staining: Consider co-staining with α-tubulin or other centrosomal markers to confirm proper localization .

• Counterstaining: Use DAPI for nuclear visualization to assess CKAP5's relationship to chromatin during mitosis .

• Confocal microscopy: Employ confocal microscopy for detailed visualization of CKAP5 at microtubule plus ends and centrosomes.

• Live cell imaging: For dynamic studies, consider CKAP5-GFP fusion proteins in combination with tubulin markers .

How can researchers effectively use CKAP5 antibodies to study mitotic spindle formation?

To effectively study mitotic spindle formation using CKAP5 antibodies:

• Cell synchronization: Use nocodazole or thymidine block to enrich for mitotic cells.

• Fixation timing: Capture cells at various mitotic stages (prophase, metaphase, anaphase, telophase).

• Co-immunostaining: Combine CKAP5 antibody with antibodies against:

  • α-tubulin to visualize the entire spindle structure

  • γ-tubulin to mark centrosomes

  • phospho-histone H3 to identify mitotic cells

  • EB1 to examine microtubule plus-end dynamics

• Cold-stable microtubule assay: Expose cells to cold treatment before fixation to assess kinetochore-attached microtubule stability.

• Quantitative analysis: Measure spindle length, width, density, and CKAP5 distribution using image analysis software .

• 3D reconstruction: Employ Z-stack imaging and 3D reconstruction to fully capture the spatial organization of CKAP5 within the mitotic spindle.

How can CKAP5 antibodies be employed in cancer research studies?

CKAP5 antibodies can be strategically used in cancer research through:

• Expression profiling: Compare CKAP5 expression levels between normal and tumor tissues, particularly in hepatomas and colonic tumors where CKAP5 is notably expressed .

• Prognostic marker analysis: Correlate CKAP5 expression with patient outcomes using tissue microarrays and IHC.

• Genetic instability assessment: Examine the relationship between CKAP5 expression and genetic instability markers, as genetically unstable cancer cell lines show selective vulnerability to CKAP5 depletion .

• Therapeutic response prediction: Evaluate whether CKAP5 expression levels correlate with response to microtubule-targeting drugs.

• Drug resistance mechanisms: Investigate CKAP5's role in chemoresistant cancer cells, such as the NCI-ADR/Res (NAR) ovarian cancer cell line that is highly sensitive to CKAP5 manipulation despite being resistant to conventional chemotherapeutics .

• Combination therapy approaches: Study how CKAP5 targeting might sensitize cancer cells to other treatments.

What are the methodological considerations for studying CKAP5 knockdown effects?

When designing experiments to study CKAP5 knockdown effects:

• siRNA design: Select siRNA sequences with confirmed efficacy and specificity for CKAP5. Multiple siRNAs should be tested to rule out off-target effects .

• Delivery method: Consider lipid nanoparticles (LNPs) for efficient delivery of siRNAs targeting CKAP5, particularly for in vivo applications .

• Knockdown verification: Confirm knockdown at both transcript level (by qPCR) and protein level (by Western blot) .

• Timing considerations: Assess phenotypes at multiple time points (48-72 hours and 6 days post-transfection) as some effects become more prominent with time .

• Cell viability assays: Use multiple methodologies (e.g., MTT, colony formation) to comprehensively evaluate viability effects .

• Mitotic analysis: Examine spindle morphology, metaphase plate formation, and chromosome segregation in CKAP5-depleted cells .

• Live-cell imaging: Track mitotic progression using fluorescent markers (tubulin-GFP, H2B-mCherry) to determine precise points of mitotic arrest or failure .

• Control selection: Include both untreated cells and cells treated with non-targeting control siRNAs .

How do researchers interpret conflicting CKAP5 antibody staining patterns?

When faced with conflicting CKAP5 antibody staining patterns:

• Epitope mapping: Determine the epitope recognized by each antibody, as different antibodies may recognize distinct domains of this large protein.

• Isoform specificity: Verify which of the three CKAP5 isoforms each antibody detects, as isoform expression may vary across tissues or cell lines .

• Cell cycle dependence: Assess whether discrepancies relate to cell cycle stage, as CKAP5 localization and modification state changes dramatically during mitosis versus interphase .

• Post-translational modifications: Consider that phosphorylation or other modifications may mask epitopes in specific cellular contexts.

• Fixation sensitivity: Test multiple fixation protocols, as some epitopes may be sensitive to particular fixatives.

• Antibody validation: Perform knockdown experiments to confirm specificity of each antibody .

• Cross-reactivity assessment: Conduct immunoprecipitation followed by mass spectrometry to identify potential cross-reactive proteins.

• Literature comparison: Compare your findings with published data, noting experimental conditions that may explain discrepancies.

How can CKAP5 antibodies be utilized in studying cancer therapeutic resistance mechanisms?

CKAP5 antibodies can provide insights into therapeutic resistance through:

• Expression correlation: Compare CKAP5 expression levels between treatment-resistant and treatment-sensitive cancer cell lines, similar to the analysis of the chemo-resistant NCI-ADR/Res (NAR) ovarian cancer cell line .

• Combination therapy assessment: Evaluate CKAP5 expression before and after treatment with conventional chemotherapeutics to identify adaptive responses.

• Patient-derived xenograft (PDX) models: Use CKAP5 antibodies to assess expression in PDX models with varying treatment responses.

• Genetic instability quantification: Combine CKAP5 antibody staining with genetic instability markers to stratify tumors for potential CKAP5-targeting therapies .

• Mitotic checkpoint analysis: Investigate whether CKAP5 expression correlates with mitotic checkpoint alterations in resistant cells.

• T-DM1 binding studies: Examine the relationship between CKAP5 expression and sensitivity to antibody-drug conjugates like T-DM1, which has been shown to bind CKAP5 .

• Comparative proteomics: Use CKAP5 immunoprecipitation followed by mass spectrometry to identify differential protein interactions in resistant versus sensitive cells.

What methodological approaches are recommended for studying CKAP5's role in microtubule dynamics?

To investigate CKAP5's role in microtubule dynamics:

How can CKAP5 antibodies be used to investigate the protein's interaction with T-DM1 and other therapeutic antibody conjugates?

To study CKAP5's interaction with antibody-drug conjugates like T-DM1:

• Co-immunoprecipitation: Use CKAP5 antibodies to pull down the protein and test for T-DM1 association, or vice versa .

• Surface plasmon resonance: Measure binding kinetics between purified CKAP5 and T-DM1 or other antibody conjugates.

• Competition assays: Determine whether pre-incubation with CKAP5 antibodies blocks T-DM1 binding to cell surfaces .

• Confocal co-localization: Assess co-localization of fluorescently labeled T-DM1 with CKAP5 antibody staining at the cell surface.

• Domain mapping: Generate truncated CKAP5 constructs to identify which domains interact with T-DM1.

• Cytotoxicity correlation: Compare T-DM1 cytotoxicity in cells with varying CKAP5 expression levels or in CKAP5 knockdown models .

• Cross-linking mass spectrometry: Identify specific contact residues between CKAP5 and T-DM1 using chemical cross-linking followed by mass spectrometry.

• Structural biology approaches: Use cryo-EM or X-ray crystallography to determine the structural basis of CKAP5-T-DM1 interactions.

What are common troubleshooting approaches for weak or non-specific CKAP5 antibody signals?

When encountering weak or non-specific CKAP5 antibody signals:

• Sample preparation optimization:

  • For Western blot: Ensure complete lysis and use fresh protease inhibitors to prevent degradation of this large protein.

  • For immunostaining: Test different fixation methods (PFA vs. methanol) as epitope accessibility may differ.

• Antibody concentration adjustment:

  • Titrate antibody concentrations to find optimal signal-to-noise ratio.

  • For Western blot: Try longer incubation times (overnight at 4°C) with more dilute antibody.

• Blocking optimization:

  • Test alternative blocking agents (BSA, normal serum, commercial blockers).

  • Extend blocking time to reduce background.

• Signal enhancement strategies:

  • For Western blot: Use high-sensitivity ECL substrates or increase exposure time.

  • For IF: Try tyramide signal amplification or higher sensitivity detection systems.

• Epitope retrieval methods:

  • For FFPE tissues: Test different antigen retrieval methods (citrate, EDTA, enzymatic).

  • For cells: Test mild detergent treatments to improve antibody accessibility.

• Secondary antibody considerations:

  • Ensure secondary antibody is compatible with primary antibody species and isotype.

  • Try fluorophore-conjugated secondary antibodies with higher brightness.

• Cross-validation:

  • Test multiple CKAP5 antibodies targeting different epitopes.

  • Confirm results with genetic approaches (siRNA knockdown) .

How should researchers interpret CKAP5 expression data in the context of genetic instability?

When interpreting CKAP5 expression in relation to genetic instability:

• Expression level correlation:

  • Compare CKAP5 expression with established genetic instability markers (chromosomal aberrations, microsatellite instability).

  • Consider that correlation does not necessarily imply causation.

• Functional consequences:

  • Assess whether CKAP5 expression levels correlate with mitotic abnormalities such as multipolar spindles or chromosome missegregation .

  • Determine if CKAP5 overexpression or knockdown affects ploidy or karyotype stability over time.

• Cell line comparisons:

  • Note that genetically unstable cancer cell lines show selective vulnerability to CKAP5 depletion, suggesting a synthetic lethal relationship .

  • Consider using a panel of cell lines with varying degrees of genetic instability to establish a correlation.

• Treatment response interpretation:

  • Elevated CKAP5 dependency in genetically unstable cells suggests potential as a therapeutic target in these contexts .

  • Determine whether CKAP5 targeting produces synthetic lethality with other genetic instability drivers.

• Mechanistic understanding:

  • Interpret CKAP5 data in context of its role in spindle formation and chromosome segregation .

  • Consider that in genetically unstable cells, proper CKAP5 function may be more critical for survival.

• Tumor heterogeneity considerations:

  • Account for intratumoral heterogeneity when assessing CKAP5 expression in patient samples.

  • Consider single-cell approaches to correlate CKAP5 expression with genetic instability at the cellular level.

What considerations are important when quantifying CKAP5 immunofluorescence signals in mitotic cells?

For accurate quantification of CKAP5 immunofluorescence in mitotic cells:

• Cell cycle staging:

  • Use specific markers (phospho-histone H3, cyclin B1) to precisely identify mitotic phases.

  • Create separate analyses for prophase, metaphase, anaphase, and telophase cells .

• Spindle morphology assessment:

  • Measure multiple parameters: spindle length, width, pole-to-pole distance, and CKAP5 distribution .

  • Quantify spindle density by measuring integrated fluorescence intensity of tubulin staining.

• Signal normalization:

  • Normalize CKAP5 signal to tubulin signal to account for variations in spindle size.

  • Include internal controls (non-mitotic cells) on the same slide for background subtraction.

• 3D consideration:

  • Collect Z-stacks to capture the entire spindle volume.

  • Use maximum intensity projections or sum slices appropriately for quantification.

• Multicentric spindle quantification:

  • Establish clear criteria for classifying normal versus abnormal spindles.

  • Quantify the percentage of cells with multicentric spindles after CKAP5 manipulation .

• Time-lapse analysis:

  • For live imaging, establish consistent exposure and acquisition parameters.

  • Track individual cells through mitosis to correlate initial CKAP5 levels with mitotic outcomes .

• Statistical considerations:

  • Analyze sufficient cell numbers (>100 per condition) to account for natural variability.

  • Apply appropriate statistical tests based on data distribution.

  • Consider biological replicates across multiple experiments.

What emerging technologies might enhance CKAP5 antibody-based research?

Emerging technologies with potential to advance CKAP5 antibody research include:

• Single-cell proteomics:

  • Apply mass cytometry (CyTOF) with CKAP5 antibodies to correlate expression with other proteins at single-cell resolution.

  • Use single-cell Western blotting to quantify CKAP5 in individual cells from heterogeneous populations.

• Advanced microscopy approaches:

  • Apply lattice light-sheet microscopy for long-term, high-resolution imaging of CKAP5 dynamics during cell division.

  • Use expansion microscopy to physically enlarge specimens for super-resolution imaging of CKAP5 at microtubule plus ends.

• Proximity labeling methods:

  • Employ APEX2 or BioID fusion proteins to identify proteins in close proximity to CKAP5 in living cells.

  • Map the dynamic CKAP5 interactome during different cell cycle stages.

• Antibody engineering:

  • Develop bifunctional antibodies targeting both CKAP5 and tubulin to study their interactions in situ.

  • Create intrabodies (intracellular antibodies) to track and potentially manipulate CKAP5 in living cells.

• CRISPR-based approaches:

  • Use CRISPR activation/inhibition to modulate CKAP5 expression while monitoring effects with antibody-based detection.

  • Create CRISPR knock-in cell lines with tagged endogenous CKAP5 for antibody-free tracking.

• Organoid and 3D culture applications:

  • Apply CKAP5 antibodies to study its role in mitotic spindle orientation in 3D organoid models.

  • Investigate how tissue architecture influences CKAP5 function and localization.

• Antibody-based proteomics:

  • Develop CKAP5 antibody arrays to screen multiple cancer types for expression patterns.

  • Combine with phospho-specific antibodies to map CKAP5 post-translational modifications.

How might CKAP5 antibody research contribute to precision oncology approaches?

CKAP5 antibody research could advance precision oncology through:

• Biomarker development:

  • Validate CKAP5 expression or localization patterns as potential biomarkers for tumor aggressiveness.

  • Develop CKAP5 antibody-based diagnostic tests to identify tumors likely to respond to mitotic inhibitors.

• Patient stratification strategies:

  • Identify patient subgroups with tumors showing high CKAP5 dependency based on genetic instability profiles .

  • Create immunohistochemistry scoring systems for CKAP5 to guide treatment decisions.

• Therapeutic monitoring:

  • Track changes in CKAP5 expression or localization during treatment as potential indicators of resistance development.

  • Use serial biopsies with CKAP5 antibody staining to monitor treatment effects on mitotic machinery.

• Combination therapy design:

  • Identify synergistic drug combinations targeting CKAP5-dependent and CKAP5-independent pathways.

  • Develop strategies that exploit synthetic lethality between CKAP5 inhibition and other genetic vulnerabilities .

• Targeted therapy development:

  • Design antibody-drug conjugates specifically targeting CKAP5 expressed on cancer cell surfaces .

  • Investigate whether CKAP5 can serve as a target for PROTAC (proteolysis targeting chimera) development.

• Resistance mechanism characterization:

  • Use CKAP5 antibodies to investigate its role in chemotherapy resistance, particularly in aggressive ovarian cancers .

  • Study how alterations in CKAP5 expression or localization contribute to treatment failure.

• Minimal residual disease detection:

  • Explore whether CKAP5 antibodies can help detect rare circulating tumor cells with specific mitotic vulnerabilities.

  • Develop multiplexed detection approaches combining CKAP5 with other cancer markers.

CKAP5 Expression and Function Across Cell Types

CKAP5 Antibody Applications and Performance Characteristics

Effects of CKAP5 Silencing on Cellular Phenotypes