ZWINT Antibody, HRP conjugated

What is ZWINT and what is its biological significance?

ZWINT (ZW10 Interactor) is a critical component of the outer kinetochore KNL1 complex that serves as a docking point for spindle assembly checkpoint components and mediates microtubule-kinetochore interactions . It plays essential roles in:

• Targeting the RZZ complex to the kinetochore during prometaphase

• Recruiting MAD2L1 to the kinetochore

• Aligning homologous chromosomes during meiotic metaphase I

• Activating spindle assembly checkpoint at unattached kinetochores

Recent studies have implicated ZWINT in cancer biology, particularly in breast cancer where its upregulation predicts poor prognosis and promotes cancer cell proliferation via cell cycle regulation .

What applications is ZWINT Antibody, HRP conjugated suitable for?

Based on available product data, ZWINT Antibody, HRP conjugated is suitable for the following applications:

The HRP conjugation eliminates the need for secondary antibody incubation, streamlining experimental workflows and potentially reducing background signal in certain applications.

What are the recommended storage conditions for ZWINT Antibody, HRP conjugated?

ZWINT Antibody, HRP conjugated should be stored at -20°C or -80°C upon receipt . Repeated freeze-thaw cycles should be avoided as they can damage antibody integrity and reduce sensitivity . The antibody is typically supplied in a stabilizing buffer containing:

• 50% Glycerol

• 0.01M PBS, pH 7.4

• 0.03% Proclin 300 as a preservative

For short-term use (within 1 month), storage at 4°C is acceptable, but long-term storage should be at -20°C or lower to maintain activity.

How should I optimize ZWINT Antibody, HRP conjugated for Western blot analysis?

For optimal Western blot results with ZWINT Antibody, HRP conjugated:

• Sample preparation: Isolate total protein using RIPA buffer with 1% PMSF protease inhibitor

• Protein denaturation: Boil samples with SDS-PAGE buffer for 5 minutes

• Membrane selection: Use PVDF membranes for optimal protein binding and signal development

• Blocking: Use 5% non-fat milk or BSA in TBST for 1 hour at room temperature

• Antibody dilution: Start with a 1:1000 dilution and adjust based on signal intensity

• Incubation conditions: Incubate with antibody solution overnight at 4°C with gentle rocking

• Washing: Perform at least 3 washes with TBST, 5-10 minutes each

• Detection: Use ECL chemiluminescence detection kit and analyze with a luminescent-image analyzer

Since the antibody is already HRP-conjugated, no secondary antibody is required, which simplifies the protocol and may reduce background.

What controls should be included when working with ZWINT Antibody, HRP conjugated?

When working with ZWINT Antibody, HRP conjugated, the following controls are essential:

Including these controls helps validate experimental findings and troubleshoot potential issues with antibody performance.

What parameters should be optimized for immunohistochemistry with ZWINT Antibody?

For immunohistochemistry applications with ZWINT Antibody, the following parameters should be optimized:

• Tissue fixation: Use formalin-fixed, paraffin-embedded tissues with appropriate fixation times to preserve epitopes

• Deparaffinization and rehydration: Process tissue sections through xylene and graded alcohol series

• Antigen retrieval: Use citrate buffer with microwave heating for optimal epitope exposure

• Endogenous peroxidase blocking: Treat with H₂O₂ to eliminate background from endogenous peroxidase activity

• Antibody dilution: Test a range of dilutions to determine optimal signal-to-noise ratio

• Incubation time and temperature: Typically overnight at 4°C or 1-2 hours at room temperature

• Detection system: Use an appropriate HRP substrate (DAB, AEC) for visualization

• Counterstaining: Apply hematoxylin for nuclear counterstaining to provide context

Optimization should be performed systematically, changing one parameter at a time and documenting results.

How can I correlate ZWINT expression with cell cycle parameters?

To correlate ZWINT expression with cell cycle parameters:

• Synchronize cells at different cell cycle phases using:

  • Double thymidine block (G1/S boundary)

  • Nocodazole treatment (M phase)

  • Serum starvation/stimulation (G0/G1)

• Monitor cell cycle distribution using flow cytometry analysis with propidium iodide staining

• Analyze ZWINT expression in synchronized populations via:

  • Western blot with ZWINT Antibody, HRP conjugated

  • Immunofluorescence to visualize subcellular localization

• Co-analyze cell cycle regulators including:

  • Cyclin D1, Cyclin E1 (G1 phase and G1/S transition markers)

  • CDK4 (cell cycle kinase)

  • CDKN1B (cell cycle inhibitor)

• Quantification and correlation:

  • Plot ZWINT expression levels against percentage of cells in each cell cycle phase

  • Perform Pearson or Spearman correlation analysis

  • Use multivariate analysis to account for additional factors

Research has shown that ZWINT promotes breast cancer proliferation via cell cycle regulation, particularly by influencing critical cell cycle regulators involved in G1 phase and G1/S transition .

What is the relationship between ZWINT and miR-204 in cancer research?

Recent research has identified ZWINT as a direct target of miR-204 in breast cancer, establishing an important regulatory axis:

• miR-204 acts as a tumor suppressor that directly targets ZWINT:

  • miR-204 binds to a specific site in the 3′-UTR of ZWINT

  • This binding inhibits ZWINT translation, reducing protein levels

• ZWINT upregulation in cancer often occurs due to:

  • Decreased miR-204 expression in cancer tissues

  • Amplification of ZWINT gene

  • Other post-transcriptional regulatory mechanisms

• Experimental validation approaches:

  • Luciferase reporter assays with wild-type and mutant ZWINT 3′-UTR

  • miR-204 mimics/inhibitors to modulate ZWINT expression

  • Western blot analysis of ZWINT protein levels after miR-204 manipulation

• Functional consequences:

  • miR-204 restoration inhibits cancer cell proliferation

  • ZWINT overexpression can rescue the effects of miR-204

  • This regulatory axis affects cell cycle progression

This miR-204/ZWINT axis represents a potential therapeutic target in breast cancer and potentially other cancer types where ZWINT is dysregulated .

How can I troubleshoot non-specific binding when using ZWINT Antibody, HRP conjugated?

When encountering non-specific binding with ZWINT Antibody, HRP conjugated, consider these troubleshooting steps:

• Antibody dilution optimization:

  • Test a titration series (e.g., 1:500, 1:1000, 1:2000)

  • Higher dilutions typically reduce non-specific binding but may decrease specific signal

• Blocking improvements:

  • Extend blocking time (1-2 hours)

  • Test alternative blocking agents (BSA, normal serum from the same species as the secondary antibody)

  • Add 0.1-0.5% BSA or 0.1% Tween-20 to antibody diluent

• Washing optimization:

  • Increase number of washes

  • Extend washing duration

  • Use fresh washing buffer with proper detergent concentration

• Sample preparation evaluation:

  • Ensure proper fixation and antigen retrieval

  • Consider different antigen retrieval methods (heat-induced vs. enzymatic)

  • Optimize incubation times and temperatures

• Endogenous enzyme activity:

  • Thoroughly block endogenous peroxidase activity

  • Use specialized blocking reagents for tissues with high endogenous peroxidase

• Antibody specificity verification:

  • Perform peptide competition assays

  • Test on known positive and negative control samples

Systematic troubleshooting should involve changing one parameter at a time and documenting results carefully .

How should ZWINT expression be quantified in tissue microarrays?

For standardized quantification of ZWINT expression in tissue microarrays (TMAs):

• Staining evaluation approach:

  • Have two independent pathologists evaluate each TMA dot

  • Use a standardized scoring system with the following components:

• Staining intensity scoring:

  • 0 (almost negative)

  • 1 (weak staining)

  • 2 (moderate staining)

  • 3 (strong staining)

• Calculation of immunoreactive score:

  • Multiply the percentage of staining region (0-100%) by intensity grade (0-3)

  • Final score ranges from 0 to 300

• Digital image analysis:

  • Consider using automated image analysis software for more objective quantification

  • Calibrate software parameters against pathologist scoring

• Statistical analysis:

  • Define appropriate cutoff values to categorize expression as "high" or "low"

  • Use receiver operating characteristic (ROC) curve analysis to determine optimal cutoff values

  • Apply these categories for survival analysis and clinical correlations

This standardized approach allows for reliable comparison across different samples and studies, facilitating meta-analyses and clinical correlations .

How can I reconcile contradictory ZWINT mRNA and protein expression data?

When facing discrepancies between ZWINT mRNA and protein expression levels:

• Consider post-transcriptional regulation:

  • miR-204 directly targets ZWINT mRNA, potentially reducing protein levels despite high mRNA expression

  • Other microRNAs may also regulate ZWINT expression

• Evaluate methodological differences:

  • mRNA detection (qPCR, RNA-seq) has different sensitivity than protein detection (Western blot, IHC)

  • Different antibodies may recognize different epitopes or isoforms

  • Sample preparation methods may affect detection efficiency

• Assess technical variables:

  • Normalization methods used for mRNA vs. protein quantification

  • Batch effects in multi-sample analyses

  • Different detection thresholds between techniques

• Biological explanations:

  • Protein stability and half-life variations

  • Temporal lag between mRNA expression and protein accumulation

  • Cell type-specific post-translational modifications affecting antibody recognition

• Integration approach:

  • Use multiple detection methods for both mRNA and protein

  • Include time course experiments to capture expression dynamics

  • Consider single-cell analyses to account for cellular heterogeneity

Understanding the full spectrum of regulatory mechanisms affecting ZWINT can help explain apparent contradictions between mRNA and protein data.

What statistical analyses are appropriate for correlating ZWINT expression with clinical outcomes?

For robust statistical analysis of ZWINT expression and clinical outcomes:

• Univariate survival analyses:

  • Kaplan-Meier curves to visualize survival differences between ZWINT-high and ZWINT-low groups

  • Log-rank tests to assess statistical significance of survival differences

  • Hazard ratios to quantify risk associated with ZWINT expression levels

• Multivariate analyses:

  • Cox proportional hazards models to determine if ZWINT is an independent prognostic factor

  • Variables to include: age, tumor size, grade, stage, molecular subtype, treatment regimen

• Correlation analyses:

  • Pearson or Spearman correlation for continuous variables

  • Chi-square tests for categorical variables

  • ANOVA for comparing ZWINT expression across multiple groups

• Predictive modeling:

  • Receiver operating characteristic (ROC) curves to assess predictive value

  • Area under the curve (AUC) calculation to quantify discriminatory power

  • Nomograms incorporating ZWINT expression with other clinical factors

• Meta-analysis approaches:

  • Forest plots to visualize effect sizes across multiple studies

  • Random-effects models to account for inter-study heterogeneity

  • Publication bias assessment using funnel plots

These statistical approaches provide a comprehensive framework for evaluating the clinical significance of ZWINT expression in cancer research .

How can ZWINT Antibody be used to study kinetochore complex assembly?

ZWINT Antibody can facilitate investigation of kinetochore complex assembly through:

• Co-immunoprecipitation studies:

  • Precipitate ZWINT and identify interacting partners

  • Compare interaction profiles across cell cycle phases

  • Identify post-translational modifications affecting complex formation

• Immunofluorescence microscopy:

  • Track ZWINT localization during mitotic progression

  • Co-stain with other kinetochore components (RZZ complex, MIS12, NDC80)

  • Quantify recruitment kinetics at the kinetochore

• Proximity ligation assays:

  • Detect direct protein-protein interactions in situ

  • Visualize spatial relationships between ZWINT and binding partners

  • Quantify interaction frequencies in different cellular contexts

• ChIP-sequencing applications:

  • Map ZWINT associations with centromeric regions

  • Identify DNA sequences involved in kinetochore assembly

  • Correlate with chromatin modifications at centromeres

• CRISPR/Cas9 edited cell lines:

  • Generate ZWINT mutants affecting specific interactions

  • Assess consequences on kinetochore assembly and function

  • Combine with live cell imaging to track dynamic processes

These approaches leverage ZWINT Antibody to elucidate the complex molecular interactions at kinetochores, which are critical for proper chromosome segregation and genome stability .

What are the considerations for multiplex immunoassays incorporating ZWINT Antibody, HRP conjugated?

When designing multiplex immunoassays that include ZWINT Antibody, HRP conjugated:

• Signal separation strategy:

  • HRP produces a chromogenic or chemiluminescent signal

  • Must be distinguishable from other detection systems in the multiplex

  • Consider chromogenic substrates with different colors for HRP vs. other enzymes

• Antibody compatibility assessment:

  • Test for cross-reactivity between antibodies in the panel

  • Ensure epitopes are accessible in multiplex conditions

  • Verify antibodies are raised in different host species to avoid cross-reactivity

• Optimization protocols:

  • Titrate each antibody individually before combining

  • Determine optimal incubation sequences

  • Test blocking reagents that work for all antibodies in the panel

• Sequential detection approach:

  • Consider detecting HRP-conjugated antibodies first

  • Implement adequate quenching before subsequent detection steps

  • Use tyramide signal amplification for enhanced sensitivity when needed

• Controls for multiplexed systems:

  • Single-stain controls to assess signal specificity

  • Minus-one controls to identify potential interference

  • Tissue microarrays with known expression patterns for validation

Careful optimization of these parameters ensures reliable results when incorporating ZWINT Antibody, HRP conjugated into multiplex immunoassays.