kiz Antibody

What is KIZ protein and why is it significant in cellular research?

KIZ (kizuna centrosomal protein) is a crucial centrosomal protein with 673 amino acid residues and a mass of approximately 75.1 kDa in its canonical form in humans. It belongs to the Kizuna protein family and plays an essential role in establishing robust mitotic centrosome architecture that can withstand forces converging on centrosomes during spindle formation. KIZ is primarily localized in the cytoplasm and exists in up to five different isoforms. This protein undergoes post-translational modifications, particularly phosphorylation, which affects its function. KIZ is conserved across multiple species, with orthologs reported in mouse, rat, bovine, frog, zebrafish, chimpanzee, and chicken, indicating its evolutionary importance in cellular processes .

What are the standard applications for KIZ antibodies in research?

KIZ antibodies are predominantly utilized in Western Blot (WB), Enzyme-Linked Immunosorbent Assay (ELISA), Immunohistochemistry (IHC), Immunofluorescence (IF), and Immunoprecipitation (IP) applications. Western Blot and ELISA are the most commonly employed techniques. For Western Blot applications, dilutions typically range from 1:500 to 1:3000, while IHC applications generally use dilutions between 1:20 and 1:200. For IF applications, recommended dilutions are usually between 1:10 and 1:100 . These applications allow researchers to detect, quantify, and visualize KIZ protein in various experimental contexts, making these antibodies valuable tools for studying centrosome biology and mitotic processes .

How can I validate KIZ antibody specificity for my experimental system?

Validation of KIZ antibodies should follow a multi-step approach:

• Positive control testing: Overexpress a tagged KIZ construct (such as c-myc tagged KIZ) in cells like COS-1. Perform dual immunostaining with both anti-KIZ and anti-tag antibodies to confirm colocalization .

• Western blot analysis: Human KIZ antibodies typically recognize a protein of approximately 100 kDa, despite the canonical form having a predicted size of 75.1 kDa. This discrepancy is attributed to the amino acid composition rather than post-translational modifications .

• Knockout/knockdown controls: If possible, use KIZ knockout or knockdown cells as negative controls to confirm antibody specificity .

• Cross-species validation: When working with non-human systems, note that human KIZ and mouse PLK1S1 share only 66% identity, making species-specific antibodies necessary for accurate detection .

• Sequential dilution assessment: Test multiple antibody concentrations to determine optimal signal-to-noise ratio for your specific application .

What molecular weight should I expect when detecting KIZ using Western blot?

When performing Western blot analysis with KIZ antibodies, you should expect to observe a band at approximately 100 kDa for human KIZ, despite its canonical form having a predicted molecular weight of 75.1 kDa. This discrepancy between predicted and observed molecular weights has been documented in multiple studies and is attributed to the unique amino acid composition of KIZ rather than post-translational modifications. In contrast, when detecting mouse PLK1S1 (the mouse ortholog of human KIZ), anti-PLK1S1 antibodies typically recognize a 76 kDa protein, which aligns closely with the predicted size of the full-length protein. These differences in observed molecular weights between species should be considered when interpreting Western blot results to avoid misidentification of your protein of interest .

What are the recommended storage conditions for KIZ antibodies to maintain activity?

For optimal preservation of KIZ antibody activity, store antibodies at -20°C in buffer containing PBS with 0.02% sodium azide and 50% glycerol at pH 7.3. Most commercially available KIZ antibodies have a shelf life of approximately 12 months when stored properly. It is critical to avoid repeated freeze/thaw cycles as these significantly diminish antibody performance. For any directly-labeled flow cytometry KIZ antibodies, storage at 2-8°C is recommended instead. When working with the antibody, aliquot the stock solution into smaller volumes upon initial thawing to minimize freeze/thaw cycles. If temporary storage is needed during experimental procedures, antibodies can be kept at 4°C for short periods (typically less than one week). Always return antibodies to proper storage conditions promptly after use to maintain their integrity and binding efficiency .

What is the optimal protocol for immunofluorescence staining of KIZ in ciliated cells?

For optimal immunofluorescence staining of KIZ in ciliated cells, follow this evidence-based protocol:

• Cell preparation: For primary fibroblasts or similar cells, induce monocilia formation by serum deprivation (24 hours in serum-free medium) .

• Fixation: Fix cells with 4% paraformaldehyde for 5 minutes at room temperature to preserve protein structure while maintaining cellular architecture .

• Permeabilization: Permeabilize with 1× PBS containing 0.2% Triton X-100 for 15 minutes to allow antibody access to intracellular structures .

• Blocking: Block with 1× PBS containing 0.1% gelatin, 0.05% Tween 20, and 0.1% bovine serum albumin (BSA) for 30-60 minutes to reduce nonspecific binding .

• Primary antibody incubation: Apply anti-KIZ antibody at 1:250 dilution in blocking buffer. For co-staining, combine with anti-acetylated-α-tubulin (1:1000) to mark ciliary structures. Incubate for 1-2 hours at room temperature or overnight at 4°C .

• Washing: Wash 3 times with PBS containing 0.05% Tween 20 for 5 minutes each.

• Secondary antibody incubation: Apply appropriate fluorophore-conjugated secondary antibodies at manufacturer-recommended dilutions for 1 hour at room temperature.

• Nuclear counterstaining: Include DAPI (1:1000) for nuclear visualization .

• Mounting: Mount using Mowiol or similar anti-fade mounting medium .

• Imaging: Capture multiple fields (approximately 10) to observe adequate numbers of cilia (about 6 per field) .

This protocol has been validated for detecting KIZ localization in human fibroblasts and can be adapted for other cell types with appropriate controls.

How can I optimize antibody pre-absorption to reduce background in KIZ immunostaining?

To effectively reduce background in KIZ immunostaining through antibody pre-absorption:

• Preparation of cellular lysates: Use untransfected cells of the same type as your experimental cells (such as COS-1 cells) to create crude cellular lysates by lysing cells in PBS with brief sonication .

• Pre-absorption ratio: Mix your primary antibody solution with the crude cell lysate at a 1:100 ratio (e.g., 1 μL lysate per 100 μL antibody solution) .

• Incubation conditions: Pre-incubate this mixture for 15 minutes at 37°C to allow non-specific antibodies to bind to cellular components present in the lysate .

• Centrifugation option: For highly sensitive experiments, centrifuge the pre-absorbed antibody mixture at 14,000 × g for 10 minutes to remove antibody-antigen complexes.

• Application to samples: Apply the pre-absorbed antibody solution to your experimental samples and proceed with your standard staining protocol .

This pre-absorption technique significantly reduces non-specific binding by allowing antibodies with off-target affinities to bind to cellular components in the lysate rather than your experimental samples. The method has been experimentally validated and is particularly useful when working with polyclonal KIZ antibodies, which typically have higher background than monoclonal antibodies.

What are the optimal dilutions for KIZ antibodies across different applications?

The following table summarizes optimal dilution ranges for KIZ antibodies across various applications based on experimental validation:

These dilution ranges serve as starting points and should be optimized for specific experimental conditions, sample types, and detection methods. Always perform a dilution series during initial validation to determine optimal signal-to-noise ratio for your specific experimental system .

How can I use KIZ antibodies to investigate centrosome abnormalities in disease models?

To effectively use KIZ antibodies for investigating centrosome abnormalities in disease models:

• Baseline characterization: First establish normal KIZ localization patterns in control samples using immunofluorescence with anti-KIZ (1:250) and centrosomal markers like anti-acetylated-α-tubulin (1:1000) .

• Quantitative parameters: Measure and quantify the following parameters using image analysis software (e.g., ImageJ):

  • Cilium length (in μm)

  • Percentage of ciliated cells

  • KIZ signal intensity at centrosomes

  • Co-localization coefficients with other centrosomal markers

• Statistical analysis: Apply appropriate statistical tests based on data distribution. For cilium length, which often follows non-normal distribution, use non-parametric tests like Mann-Whitney U test comparing medians. For percentage of ciliated cells, use chi-square tests comparing categorical variables .

• Mutation impact assessment: When investigating disease-causing mutations, implement serum deprivation protocols (24 hours) to induce ciliogenesis, allowing visualization of potential structural abnormalities. Compare mutant cells with wild-type controls using the quantitative parameters above .

• Protein expression analysis: Complement immunostaining with Western blot analysis to detect potential alterations in KIZ protein levels or molecular weight shifts that might indicate post-translational modifications or truncations in disease models .

This approach has been successfully implemented in studies examining the impact of KIZ mutations in retinal ciliopathies, where compound heterozygous mutations (e.g., c.52G>T and c.119_122del) were shown to affect ciliary structure and function .

What approaches can help differentiate between KIZ isoforms in experimental systems?

Differentiating between KIZ isoforms requires a multi-faceted approach:

• Isoform-specific antibody selection: KIZ has up to five reported isoforms. When selecting antibodies, verify the immunogen sequence against known isoform-specific regions. Commercial antibodies like those from FineTest (FNab01082) target specific regions (e.g., amino acids 187-438) that may be present in some but not all isoforms .

• Western blot resolution optimization: Use gradient gels (4-15% acrylamide) to achieve better separation of closely sized isoforms. Extended run times at lower voltages improve resolution between bands that may differ by only a few kilodaltons.

• Isoform-specific primers: Complement antibody-based detection with RT-PCR using primers that span isoform-specific exon junctions.

• Overexpression controls: Generate expression constructs for individual KIZ isoforms with distinguishable tags (e.g., c-myc, FLAG, or GFP) to create positive controls for each isoform. These can be run alongside experimental samples to identify specific isoform bands .

• Phosphatase treatment: Since KIZ undergoes phosphorylation, treat protein samples with lambda phosphatase before Western blot analysis to eliminate mobility shifts caused by differential phosphorylation states that might be confused with isoform differences .

• Mass spectrometry validation: For definitive isoform identification, excise bands of interest and perform mass spectrometry to identify isoform-specific peptide sequences.

These combined approaches provide robust identification of specific KIZ isoforms in experimental systems, essential for understanding isoform-specific functions in different cellular contexts.

How do mutations in KIZ affect antibody recognition and experimental design?

Mutations in KIZ can significantly impact antibody recognition, necessitating careful experimental design considerations:

• Epitope mapping: KIZ mutations, particularly truncating mutations like c.52G>T (p.Glu18*) and c.119_122del (p.Lys40Ilefs14), can result in loss of epitopes. When studying samples with known mutations, verify whether the antibody's target epitope is preserved in the mutant protein. For instance, antibodies targeting regions beyond amino acid position 40 would fail to detect the truncated protein resulting from the p.Lys40Ilefs14 mutation .

• Validation strategy: In samples with KIZ mutations, validate antibody performance using multiple antibodies targeting different epitopes to ensure comprehensive detection of potentially truncated proteins .

• Control selection: When investigating mutations, use appropriate positive controls such as:

  • Wild-type cells expressing full-length KIZ

  • Cells expressing the specific mutant form of KIZ

  • Overexpression systems with tagged constructs containing the same mutation

• Detection method adaptations: For mutations resulting in significantly altered protein size, adjust gel percentage and running conditions in Western blot protocols to optimize detection of smaller fragments.

• Combined methodologies: Complement protein detection with mRNA analysis to confirm whether mutations affect transcript stability, which could result in absence of protein independent of antibody recognition issues .

This comprehensive approach ensures accurate interpretation of results when studying KIZ mutations, preventing false negatives due to epitope loss rather than actual protein absence.

What are the considerations for cross-species applications of KIZ antibodies?

When applying KIZ antibodies across different species, several critical factors must be considered:

• Sequence homology assessment: Human KIZ and its mouse ortholog PLK1S1 share only 66% amino acid identity, which significantly impacts antibody cross-reactivity. Before using a KIZ antibody across species, perform sequence alignment of the immunogen region against the target species sequence to predict potential cross-reactivity .

• Species-specific antibody selection: Choose antibodies raised against the species you're studying whenever possible. For example, the anti-PLK1S1 antibody specifically recognizes the mouse 76 kDa protein, while human anti-KIZ antibodies typically detect a 100 kDa protein .

• Validation requirements: When using antibodies across species, additional validation steps are essential:

  • Western blot analysis comparing target species with positive control species

  • Immunofluorescence with known localization patterns

  • Pre-absorption with peptides from both species to compare specificity

• Adjusted detection parameters: Differences in protein size between species (e.g., 76 kDa in mouse vs. 100 kDa in humans) necessitate adjusted gel concentrations and running conditions in Western blot applications .

• Differential dilution optimization: Cross-species applications often require different antibody dilutions than those recommended for the original target species. Generally, higher concentrations (2-3× higher) are needed for cross-species applications to compensate for reduced affinity.

These considerations are particularly important when studying KIZ across evolutionary diverse models, as significant structural differences exist despite functional conservation .

How can I address weak or absent KIZ signal in Western blot applications?

When encountering weak or absent KIZ signal in Western blot applications, implement this systematic troubleshooting approach:

• Sample preparation optimization:

  • Ensure complete cell lysis using buffer containing 50 mM Tris pH 7.5, 150 mM NaCl, 1% Triton-X100

  • Include sonication (three 10-second bursts) during protein extraction to improve release of centrosome-associated proteins

  • For challenging samples, consider using stronger lysis conditions with brief vortexing

• Protein loading adjustments:

  • Increase protein loading to at least 20 μg per lane

  • For low-expression samples, consider immunoprecipitation before Western blot

• Transfer optimization:

  • For the 100 kDa KIZ protein, extend transfer time by 25-50% compared to standard protocols

  • Consider using reduced methanol concentration (5-10%) in transfer buffer for improved transfer of larger proteins

• Antibody concentration:

  • Reduce dilution to 1:500 for weak signals

  • Extend primary antibody incubation to overnight at 4°C

• Detection enhancement:

  • Use high-sensitivity ECL substrate for chemiluminescence detection

  • Extend exposure time incrementally up to 30 minutes

  • Consider signal amplification systems like biotin-streptavidin for very low signals

• Control verification:

  • Run positive control of overexpressed KIZ in parallel to confirm antibody functionality

  • Verify detection of housekeeping proteins to confirm successful transfer

This systematic approach addresses the most common causes of weak or absent KIZ signal in Western blot applications, particularly important when working with low-abundance centrosomal proteins like KIZ.

What factors affect cilium length measurements when using KIZ antibodies?

Accurate cilium length measurement using KIZ antibodies requires careful consideration of several experimental factors:

• Fixation impact: Different fixation methods significantly affect ciliary structure measurement. Use 4% paraformaldehyde for 5 minutes to maintain structural integrity while allowing antibody penetration .

• Serum deprivation standardization: The duration of serum deprivation directly affects cilium length. Standardize to 24 hours of serum deprivation across all experimental groups to ensure comparable ciliogenesis conditions .

• Antibody selection considerations: When measuring cilium length, combine KIZ antibody (1:250) with established ciliary markers like acetylated-α-tubulin (1:1000) to clearly demarcate the entire ciliary structure .

• Image acquisition parameters: Use consistent confocal settings (laser power, gain, pinhole, z-stack intervals) across all samples. Capture multiple fields (minimum 10) with approximately 6 cilia per field to ensure statistical robustness .

• Measurement methodology standardization: When using ImageJ for length measurement, standardize the methodology:

  • Measure along the curved path of the cilium rather than straight-line distance

  • Use the same channel (typically acetylated-α-tubulin) for all measurements

  • Apply consistent thresholding parameters across all images

• Statistical analysis approach: Due to non-normal distribution of cilium length data, use non-parametric statistical tests (e.g., Mann-Whitney U test) that compare medians rather than means. Present data in box plot format showing median, quartiles, and maximum/minimum values .

Following these methodological considerations ensures accurate and reproducible cilium length measurements when using KIZ antibodies in ciliopathy research.

How should conflicting results between different KIZ antibodies be interpreted?

When faced with conflicting results between different KIZ antibodies, implement this systematic interpretation framework:

• Epitope-based analysis: Different antibodies target distinct epitopes within the KIZ protein. Map the epitope regions of each antibody (e.g., amino acids 187-438 for some commercial antibodies) and assess whether known mutations, splice variants, or post-translational modifications might affect epitope accessibility in your experimental system .

• Clonality comparison: Polyclonal antibodies (like FNab01082) typically recognize multiple epitopes while monoclonal antibodies target single epitopes. Conflicting results may reflect detection of different KIZ isoforms or post-translationally modified variants .

• Validation hierarchy establishment: Prioritize results from antibodies with the most extensive validation evidence:

  • Those validated in knockout/knockdown systems

  • Those with demonstrated specificity in overexpression models

  • Those with published validation data in similar experimental contexts

• Cross-methodology verification: When antibodies show conflicting results in one application (e.g., Western blot), test in alternative applications (e.g., immunofluorescence) to determine if the conflict is application-specific.

• Blocking peptide competition: Perform competition assays with specific peptides matching the immunogen sequences to determine which antibody shows highest specificity.

• Integrated interpretation approach: Rather than choosing one antibody as "correct," consider that different antibodies may reveal complementary aspects of KIZ biology, particularly given its multiple isoforms and post-translational modifications .

This framework allows researchers to extract meaningful biological insights from seemingly conflicting antibody results, potentially revealing important aspects of KIZ regulation that would be missed by relying on a single antibody.

How can KIZ antibodies contribute to understanding ciliopathy disease mechanisms?

KIZ antibodies offer powerful tools for investigating ciliopathy disease mechanisms through multiple research approaches:

• Mutation-phenotype correlation studies: KIZ antibodies enable detailed characterization of cellular consequences of different mutations. For instance, in studies of retinal ciliopathies, KIZ antibodies have revealed that compound heterozygous mutations (c.52G>T and c.119_122del) affect ciliary structure and function. Similar approaches can be extended to other ciliopathies to establish genotype-phenotype correlations .

• Quantitative centrosome/cilium analysis: By combining anti-KIZ (1:250) with ciliary markers like acetylated-α-tubulin (1:1000), researchers can quantitatively assess:

  • Changes in cilium length

  • Alterations in percentage of ciliated cells

  • Modifications in centrosome architecture

  • Disruptions in ciliary protein localization patterns

• Protein interaction network mapping: Using KIZ antibodies for co-immunoprecipitation studies can identify interaction partners that may be disrupted in disease states, revealing pathways contributing to ciliopathies.

• Post-translational modification profiling: KIZ undergoes phosphorylation, which can be studied using phospho-specific antibodies to determine how disease mutations affect regulatory modifications .

• Therapeutic screening platforms: KIZ antibodies can be used to develop high-content screening assays for compounds that rescue ciliary defects in patient-derived cells, potentially identifying therapeutic candidates for ciliopathies.

By implementing these approaches, researchers can leverage KIZ antibodies to establish mechanistic connections between genetic mutations and cellular phenotypes in ciliopathies, potentially revealing novel therapeutic targets for these currently incurable disorders.

What are the emerging applications for KIZ antibodies in cancer research?

KIZ antibodies are increasingly valuable in cancer research due to the protein's critical role in centrosome integrity and mitotic spindle formation:

• Centrosome amplification assessment: As a centrosomal protein required for robust mitotic centrosome architecture, KIZ antibodies can be used to evaluate centrosome amplification—a hallmark of many cancers. By quantifying KIZ-positive centrosomes per cell, researchers can detect abnormalities associated with genomic instability .

• Cell division error monitoring: KIZ is essential for withstanding forces that converge on centrosomes during spindle formation. Using KIZ antibodies in time-lapse immunofluorescence, researchers can track centrosome integrity during mitosis in cancer cells, potentially identifying vulnerabilities in the mitotic machinery that could be therapeutically targeted.

• Chemotherapy response biomarker development: Since KIZ interacts with PLK1 (as suggested by its alternative name PLK1S1), antibodies against KIZ can help evaluate potential resistance mechanisms to PLK1 inhibitors, an emerging class of anti-cancer drugs .

• Cell cycle checkpoint analysis: KIZ antibodies can be combined with cell cycle markers to investigate how centrosome abnormalities affect checkpoint activation in cancer cells, potentially revealing mechanisms of cell cycle dysregulation.

• Therapeutic target validation: As centrosome clustering is a survival strategy for cancer cells with supernumerary centrosomes, KIZ antibodies can help validate the importance of this protein in maintaining viable mitosis in cancer cells, potentially identifying it as a therapeutic target.

These emerging applications position KIZ antibodies as valuable tools in understanding the role of centrosome abnormalities in cancer development and progression, potentially leading to new diagnostic or therapeutic approaches.

What buffer system modifications can improve KIZ antibody performance?

Optimizing buffer systems can significantly enhance KIZ antibody performance across applications:

• Storage buffer optimization: The standard buffer for KIZ antibodies contains PBS with 0.02% sodium azide and 50% glycerol at pH 7.3. For improved long-term stability (beyond 12 months), consider these modifications:

  • Increase glycerol concentration to 60-65% for antibodies stored at -20°C

  • Add 1% BSA as an additional stabilizing protein

  • Consider alternative preservatives like Proclin300 if sodium azide interferes with downstream applications

• Blocking buffer enhancements: For immunostaining applications, optimize blocking with:

  • 1× PBS containing 0.1% gelatin, 0.05% Tween 20, and 0.1% BSA to reduce background

  • Pre-incubate primary antibody solution with crude cell lysate (1:100) at 37°C for 15 minutes to remove non-specific binding

• Lysis buffer modifications: For protein extraction prior to Western blot:

  • Standard buffer containing 50 mM Tris pH 7.5, 150 mM NaCl, 1% Triton-X100

  • Include phosphatase inhibitors to preserve phosphorylation status

  • Consider sonication (three 10-second bursts) to improve centrosomal protein release

• Antigen retrieval buffer optimization: For IHC applications on fixed tissues:

  • Test both citrate buffer (pH 6.0) and Tris-EDTA (pH 9.0) to identify optimal epitope unmasking conditions

  • Extended incubation at 95°C may be necessary for complete retrieval of centrosomal epitopes

• Elution buffer considerations: For immunoprecipitation applications:

  • Gentle elution with 0.1M glycine (pH 2.5) followed by immediate neutralization

  • Alternative elution with 8M urea for more stringent conditions

These buffer system modifications have been developed through experimental optimization and can significantly improve detection sensitivity, reduce background, and enhance reproducibility across KIZ antibody applications.

How can statistical analysis be optimized for cilium studies using KIZ antibodies?

Optimizing statistical analysis for cilium studies using KIZ antibodies requires consideration of both biological variability and non-normal data distribution:

• Appropriate statistical test selection: Since cilium length data typically follows a non-normal distribution, employ non-parametric tests like the Mann-Whitney U test that compare medians rather than means. For categorical data like the percentage of ciliated cells, chi-square tests are appropriate .

• Sampling strategy optimization: To achieve robust statistical power:

  • Capture at least 10 fields per sample

  • Analyze approximately 6 cilia per field (minimum 60 cilia total)

  • Ensure equal division between control and experimental groups (e.g., 30 control and 30 affected cells)

• Data presentation refinement: Present cilium length data in box plot format showing:

  • Median (central bar)

  • Quartiles (box boundaries)

  • Maximum and minimum values (whiskers)

  • Individual data points for small sample sizes

• Power analysis implementation: Perform power analysis before experiments to determine minimum sample sizes needed to detect biologically relevant differences in cilium length or percentage of ciliated cells.

• Multiple comparison correction: When analyzing multiple parameters (e.g., cilium length, percentage of ciliated cells, KIZ localization), apply appropriate corrections like Bonferroni or False Discovery Rate to maintain appropriate family-wise error rates.

• Paired analysis consideration: When comparing the same cells under different conditions or time points, use paired tests (e.g., Wilcoxon signed-rank test) to increase statistical power.

This optimized statistical approach ensures that subtle but biologically significant changes in ciliary parameters are accurately detected and interpreted in KIZ-related research, improving reproducibility and translational relevance of findings .