ZWINT Antibody, FITC conjugated

What is ZWINT and what is its primary cellular function?

ZWINT (ZW10 Interacting Protein) serves as a critical component of the outer kinetochore KNL1 complex that mediates microtubule-kinetochore interactions during cell division. It functions as a docking point for spindle assembly checkpoint components . ZWINT plays a crucial role in chromosome segregation by helping to establish the physical connection between centromeric DNA and spindle microtubules, thereby ensuring genomic stability . The protein works closely with other kinetochore components such as ZW10 and Rod to monitor and regulate the attachment of spindle microtubules, preventing aneuploidy during cell division .

How does ZWINT interact with other kinetochore proteins?

ZWINT interacts with multiple protein complexes at the kinetochore. Notably, it resides in separate complexes from its binding partner ZW10. While ZW10 exists in a complex with Rod and Zwilch, ZWINT is part of a distinct complex of structural kinetochore components that includes Mis12 and Ndc80-Hec1 . Despite this separation, ZWINT serves as a critical linker between these complexes. Immunoprecipitation studies have shown that a small but significant fraction of ZW10 remains associated with ZWINT under experimental conditions . This interaction is functionally important, as ZWINT is essential for recruiting the ZW10 complex to unattached kinetochores during mitosis .

What happens when ZWINT function is disrupted in cells?

Disruption of ZWINT function leads to severe mitotic defects due to its essential role in the spindle assembly checkpoint. When ZWINT is depleted (to approximately 15% of normal levels), ZW10 fails to localize to kinetochores . This disruption compromises mitotic checkpoint signaling, as demonstrated by flow cytometry measurements of phospho-histone H3 levels. While control cells show a 10-fold increase in mitotic index after nocodazole treatment, ZWINT-depleted cells show only a 2-fold increase, indicating a severely compromised checkpoint response . This failure in checkpoint function can lead to chromosomal instability, aneuploidy, and potentially contribute to cancer development.

What types of ZWINT antibodies are available for research applications?

Primary ZWINT antibodies are available in several formats, with polyclonal rabbit antibodies being commonly used in research. For example, the rabbit polyclonal ZWINT antibody (ab71982) has been validated for immunoprecipitation (IP), western blotting (WB), and immunocytochemistry/immunofluorescence (ICC/IF) applications with human samples . When selecting a ZWINT antibody, researchers should consider:

• Host species (rabbit antibodies are common for ZWINT detection)

• Clonality (polyclonal vs. monoclonal)

• Validated applications (IP, WB, ICC/IF, etc.)

• Species reactivity (most are validated against human ZWINT)

• Immunogen information (synthetic peptide vs. recombinant protein)

The choice between these options depends on the specific experimental requirements and detection methods planned.

How do FITC-conjugated detection systems work with ZWINT antibodies?

FITC (Fluorescein isothiocyanate) conjugation provides a direct fluorescent detection method for visualizing ZWINT localization. Researchers typically employ one of two approaches:

• Direct conjugation: Primary ZWINT antibodies directly conjugated to FITC

• Indirect detection: Unconjugated primary ZWINT antibodies followed by FITC-conjugated secondary antibodies

The indirect method is more commonly used in research settings. For example, in studies examining ZW10's role in mitotic checkpoint signaling, researchers incubated samples with primary rabbit antibodies against ZWINT followed by FITC-conjugated donkey-anti-rabbit secondary antibodies for one hour on ice and in darkness to preserve fluorescence . This indirect approach offers amplification of signal through multiple secondary antibody binding, enhancing detection sensitivity.

What controls should be included when using ZWINT antibodies with FITC detection?

Proper experimental controls are essential when using ZWINT antibodies with FITC detection:

These controls help distinguish true ZWINT signals from background fluorescence and confirm proper functioning of both primary and FITC-conjugated secondary antibodies.

What are the optimal fixation and permeabilization conditions for ZWINT immunofluorescence?

The selection of fixation and permeabilization methods significantly impacts ZWINT detection quality:

For optimal ZWINT immunofluorescence at kinetochores:

• Preferred fixation: 4% paraformaldehyde for 10-15 minutes at room temperature preserves protein epitopes while maintaining cellular architecture

• Alternative fixation: Methanol fixation (-20°C for 10 minutes) can sometimes provide superior results for nuclear/kinetochore antigens

• Permeabilization: 0.1-0.5% Triton X-100 for 5-10 minutes is generally effective

• Blocking: 3-5% BSA or normal serum from the same species as the secondary antibody

Researchers should note that over-fixation can mask epitopes, while insufficient permeabilization may prevent antibody access to nuclear structures where ZWINT localizes during mitosis. Optimization for specific cell types may be necessary, particularly when working with different cancer cell lines that may display altered nuclear architecture .

How should primary and FITC-conjugated secondary antibodies be optimized for ZWINT detection?

Optimization of antibody concentrations and incubation conditions is critical for specific ZWINT detection:

• Primary antibody dilution: Typically 1:100 to 1:500 depending on antibody concentration and affinity

• Incubation conditions: Best results often achieved with overnight incubation at 4°C in a humidified chamber

• FITC-conjugated secondary antibody: Usually effective at 1:200 to 1:1000 dilution

• Secondary antibody incubation: One hour at room temperature in darkness

• Washing steps: At least 3-5 washes with PBS + 0.1% Tween-20 between antibody incubations

Titration experiments should be performed to determine optimal concentrations that maximize signal-to-noise ratio. For co-localization studies, careful selection of fluorophores with minimal spectral overlap is essential when ZWINT detection needs to be combined with other kinetochore markers.

What image acquisition parameters are critical for accurate ZWINT localization analysis?

Proper image acquisition ensures accurate representation of ZWINT localization patterns:

• Microscope type: Confocal microscopy is preferred for precise localization at kinetochores

• Objective: 63× or 100× oil immersion objectives recommended for kinetochore resolution

• Z-stack acquisition: Essential for capturing three-dimensional distribution of ZWINT at kinetochores

• Exposure settings: Determine optimal exposure to avoid saturation while capturing dynamics of expression

• Pinhole settings: Adjust to achieve optical sectioning appropriate for kinetochore structures

• Signal-to-noise optimization: Use appropriate filters and detector settings to minimize autofluorescence

How can ZWINT antibodies be used to study mitotic checkpoint regulation?

ZWINT antibodies are valuable tools for investigating the spindle assembly checkpoint mechanism:

The mitotic checkpoint ensures proper chromosome segregation, and ZWINT plays a key role by linking structural kinetochore components with checkpoint signaling proteins. Research has shown that ZWINT is essential for recruiting the ZW10 complex to kinetochores, which in turn is required for stable binding of the Mad1-Mad2 complex to unattached kinetochores . This cascade ultimately generates the "stop anaphase" inhibitor signal.

Methodological approaches include:

• Immunofluorescence with FITC detection to visualize ZWINT localization during different mitotic phases

• Co-localization studies with other checkpoint proteins (Mad1, Mad2, BubR1)

• Examination of ZWINT dynamics following treatment with spindle poisons like nocodazole

• Correlating ZWINT localization patterns with chromosome segregation errors

These approaches have revealed that ZWINT depletion severely compromises mitotic checkpoint signaling, with cells showing only a 2-fold increase in mitotic index after nocodazole treatment compared to the 10-fold increase seen in control cells .

What are the implications of ZWINT in cancer research, and how can antibodies help study this connection?

ZWINT overexpression has been observed in multiple cancer types, making it an important research target:

Bioinformatic and tissue array chip analyses have indicated ZWINT overexpression in pancreatic cancer, with expression levels further induced under hypoxic conditions . Similar patterns have been observed in glioblastoma cells. ZWINT appears to promote cancer cell proliferation and cell cycle progression, suggesting its potential as a therapeutic target.

Research methodologies include:

• Immunohistochemistry with ZWINT antibodies to assess expression in cancer tissues

• Quantitative analysis of ZWINT levels in hypoxic versus normoxic conditions

• Correlation of ZWINT expression with tumor grade and patient outcomes

• Investigation of ZWINT's role in p53/p21 signaling pathways in cancer cells

For example, CHIP assays can evaluate HIF1α interaction with the ZWINT promoter under hypoxic conditions, while immunoprecipitation and immunofluorescence can examine interactions between ZWINT, MDM2, and p53 in cancer cells .

How can ZWINT antibodies be used to investigate chromosomal instability mechanisms?

ZWINT's role in ensuring proper chromosome segregation makes it valuable for studying chromosomal instability:

Methodological approaches include:

• Combined ZWINT immunofluorescence with fluorescence in situ hybridization (FISH) to simultaneously visualize kinetochore function and chromosome positioning

• Live-cell imaging with fluorescently tagged ZWINT to monitor dynamics during chromosome segregation

• Analysis of aneuploidy rates following ZWINT depletion or overexpression

• Investigation of ZWINT's interactions with structural kinetochore components like Mis12 and Ndc80-Hec1

Research has demonstrated that disrupting ZWINT function impairs the spindle assembly checkpoint, potentially leading to chromosomal instability. For instance, when ZWINT is depleted, ZW10 fails to localize to kinetochores, compromising the checkpoint that prevents aneuploidy during cell division .

How can ZWINT antibodies be used to study cell cycle-specific protein interactions?

ZWINT demonstrates phase-specific localization and interactions during the cell cycle:

Methodological approaches:

• Synchronization strategies:

  • Double thymidine block for G1/S transition

  • Nocodazole treatment for prometaphase arrest

  • RO-3306 for G2 arrest

• Co-immunoprecipitation techniques:

  • Lysates from synchronized cell populations

  • ZWINT antibodies for pull-down followed by detection of interacting partners

  • Reciprocal IP with antibodies against suspected interacting proteins

• Proximity ligation assays:

  • Visualization of ZWINT protein interactions in situ

  • Quantification of interaction frequency during different cell cycle phases

These approaches have revealed that ZWINT forms distinct complexes throughout the cell cycle. For example, ZWINT associates with structural kinetochore components including Mis12, Ndc80-HEC1, Spc24, and AF15q14, while its binding partner ZW10 exists in a separate complex with Rod and Zwilch . These differential interactions appear critical for proper cell cycle progression.

What cell cycle defects are associated with ZWINT dysfunction, and how can they be measured?

ZWINT dysfunction leads to specific cell cycle abnormalities that can be quantitatively assessed:

Observed defects include:

• Compromised mitotic checkpoint signaling

• Aberrant kinetochore-microtubule attachments

• Chromosome segregation errors

• Disrupted cell cycle progression

Quantitative assessment methods:

• Flow cytometry: Measuring cell cycle distribution after BrdU incorporation reveals that ZWINT disruption affects G2/M progression. For example, in U87-MG and DBTRG-05 cells treated with camptothecin (which affects the cell cycle), a considerable G2/M arrest was observed (U87 72h/U87 NT: 27.9%/19.6%; DBTRG 72h/DBTRG NT: 33.8%/12.6%) with increased non-cycling S-phase cells .

• Time-lapse microscopy: Tracking mitotic duration and chromosome movements

• Immunofluorescence: Quantifying lagging chromosomes and micronuclei formation

• Gene expression analysis: Evaluating downstream effects on cell cycle regulators

Research indicates that ZWINT dysfunction specifically impacts the spindle assembly checkpoint pathway, with inhibition of BUB1, BUB3, and MAD2 mRNAs observed in senescent U87-MG cells .

What is the relationship between ZWINT and DNA damage response pathways?

ZWINT's function intersects with DNA damage response mechanisms:

Microarray analyses of cells treated with camptothecin (a topoisomerase I inhibitor that induces DNA damage) have revealed coordinated changes in gene expression involving both ZWINT and DNA damage response pathways . Functional analysis using High-Throughput GoMINER and EASE tools highlighted that down-regulation of cell cycle and mitosis genes (including ZWINT-related pathways) occurred alongside up-regulation of cell growth inhibition and DNA damage response genes .

Experimental approaches to study this relationship include:

• Immunofluorescence co-localization of ZWINT with γH2AX foci after DNA damage

• Analysis of ZWINT phosphorylation status following genotoxic stress

• Evaluation of ZWINT levels and localization in cells with compromised DNA repair machinery

• Chromosome spread analysis to assess structural abnormalities when ZWINT function is impaired

These studies suggest that ZWINT may function as an integration point between kinetochore attachment sensing and DNA damage response pathways, potentially explaining its involvement in both cancer progression and treatment response.

What are common problems encountered with ZWINT immunofluorescence, and how can they be resolved?

Researchers may encounter several challenges when performing ZWINT immunofluorescence:

When troubleshooting, remember that ZWINT localization is highly cell cycle-dependent, with strong kinetochore signals primarily visible during prometaphase. In unsynchronized populations, only a small percentage of cells will show clear kinetochore localization.

How can researchers optimize ZWINT antibody specificity for challenging applications?

Enhancing antibody specificity for ZWINT detection:

• Antibody validation strategies:

  • siRNA/shRNA ZWINT knockdown as negative controls

  • Peptide competition assays to confirm epitope specificity

  • Western blot validation before immunofluorescence applications

  • Testing multiple ZWINT antibody clones targeting different epitopes

• Signal enhancement approaches:

  • Tyramide signal amplification for low abundance detection

  • Optimized antigen retrieval for fixed samples

  • Use of high-sensitivity detection systems

• Background reduction techniques:

  • Pre-adsorption of antibodies with acetone powder from non-expressing tissues

  • Extended blocking with serum plus BSA combinations

  • Inclusion of non-ionic detergents in antibody diluents

These optimization approaches are particularly important when studying ZWINT in complex samples or when examining subtle changes in ZWINT localization patterns during different cellular states.

What considerations are important when quantifying ZWINT expression or localization?

Accurate quantification of ZWINT requires careful methodological considerations:

• Normalization strategies:

  • Internal loading controls for Western blot (housekeeping proteins)

  • Reference genes for qRT-PCR (validated through stability testing)

  • Cell-by-cell normalization for immunofluorescence intensity

• Cell cycle considerations:

  • Account for cell cycle-dependent expression/localization

  • Use cell cycle markers (pH3, PCNA, cyclin B) for subpopulation analysis

  • Consider synchronization for homogeneous populations

• Statistical approaches:

  • Determine appropriate sample sizes through power analysis

  • Apply nonparametric statistics for heterogeneous distributions

  • Use mixed-effects models for experiments with multiple sources of variation

• Image analysis parameters:

  • Define consistent thresholding methods for signal quantification

  • Implement automated analysis pipelines to reduce bias

  • Include spatial distribution metrics beyond simple intensity measurements

For example, in microarray analysis of ZWINT expression changes, researchers have employed MAANOVA (MicroArray ANalysis Of VAriance) with appropriate statistical filtering to identify significant changes in gene expression during time-course treatments .

How can ZWINT antibodies be combined with live-cell imaging techniques?

Integrating ZWINT antibody approaches with live-cell imaging:

While traditional antibody-based detection requires fixed cells, researchers can combine fixed and live approaches to gain comprehensive insights:

• Sequential live-fixed imaging:

  • Track live cells expressing fluorescent markers (e.g., H2B-GFP)

  • Fix at specific time points or after observed events

  • Perform ZWINT immunofluorescence with FITC or other compatible fluorophores

  • Relocate previously imaged cells for correlative analysis

• Antibody fragment approaches:

  • Generate Fab fragments from ZWINT antibodies

  • Fluorescently label fragments for live-cell introduction

  • Use cell-penetrating peptides or microinjection for delivery

• Complementary marker strategies:

  • Express fluorescently-tagged binding partners of ZWINT in live cells

  • Correlate live dynamics with fixed ZWINT localization

  • Use inducible expression systems to avoid artifacts

These approaches can reveal how ZWINT dynamics correlate with cellular events such as checkpoint activation, chromosome alignment, and anaphase onset.

What multi-omics approaches can be combined with ZWINT antibody studies?

Integrating ZWINT antibody techniques with broader omics approaches provides comprehensive insights:

• Proteomics integration:

  • Immunoprecipitation with ZWINT antibodies followed by mass spectrometry

  • Identification of cell cycle-specific interaction networks

  • Phosphoproteomic analysis of ZWINT and associated proteins

• Transcriptomics correlation:

  • Combine ZWINT protein levels with gene expression profiles

  • Identify transcriptional networks affected by ZWINT disruption

  • Compare normal vs. cancer tissues for ZWINT-associated signatures

• Chromatin association studies:

  • ChIP-seq with ZWINT-interacting transcription factors

  • Analysis of chromosome structural abnormalities following ZWINT depletion

  • Integration with Hi-C data to assess chromosome territory organization

Multi-omics approaches have revealed that ZWINT function extends beyond its structural role at kinetochores. For example, gene expression analysis in camptothecin-treated glioblastoma cells demonstrated that ZWINT disruption affects pathways including DNA metabolism, mitosis regulation, and inflammation response .

How can ZWINT antibodies contribute to understanding cancer therapeutic responses?

ZWINT's emerging role in cancer biology presents opportunities for therapeutic research:

• Predictive biomarker potential:

  • Quantitative assessment of ZWINT expression in patient samples

  • Correlation with treatment outcomes for specific therapeutic modalities

  • Development of standardized ZWINT immunohistochemistry protocols

• Therapeutic targeting approaches:

  • Antibody-based disruption of ZWINT interactions

  • Small molecule screening using ZWINT immunofluorescence as readout

  • Synthetic lethality screening in ZWINT-overexpressing cancer cells

• Resistance mechanism studies:

  • Analysis of ZWINT expression in treatment-resistant cells

  • Investigation of ZWINT's role in hypoxia-induced treatment resistance

  • Combined targeting of ZWINT and DNA damage response pathways

Research has demonstrated that ZWINT is overexpressed in pancreatic cancer and induced under hypoxic conditions, where it promotes cancer cell proliferation and cell cycle progression . Additionally, ZWINT has been implicated in camptothecin resistance mechanisms in glioblastoma cells , suggesting its potential importance in chemotherapy response.