KNSTRN Antibody

What is KNSTRN and what is its biological function?

KNSTRN (Kinetochore-Localized Astrin/SPAG5 Binding Protein) is an essential component of the mitotic spindle required for faithful chromosome segregation and progression into anaphase. It promotes the metaphase-to-anaphase transition and is required for chromosome alignment, normal timing of sister chromatid segregation, and maintenance of spindle pole architecture . The astrin (SPAG5)-kinastrin (SKAP) complex promotes stable microtubule-kinetochore attachments . KNSTRN is primarily localized in the nucleus and cytoplasm, with a canonical protein length of 316 amino acid residues and a mass of approximately 35.4 kDa in humans .

What are the common applications for KNSTRN antibodies in research?

KNSTRN antibodies are primarily used for immunodetection of the kinetochore localized astrin (SPAG5) binding protein. The most common applications include:

• Western Blot (WB): For detecting and quantifying KNSTRN protein expression in cell or tissue lysates

• Immunocytochemistry (ICC): For visualizing subcellular localization in cultured cells

• Immunohistochemistry (IHC): For detecting KNSTRN expression in tissue sections

These applications are essential for studying KNSTRN's role in mitotic spindle function, chromosome segregation, and various disease contexts.

What are the different types of KNSTRN antibodies available for research?

Researchers can select from several types of KNSTRN antibodies based on their experimental needs:

• Host species: Primarily rabbit polyclonal antibodies

• Target regions: Antibodies targeting different epitopes including N-terminal region, middle region, and specific amino acid sequences (e.g., AA 31-80, AA 151-200)

• Reactivity: Antibodies with varying species reactivity including human-specific and those cross-reactive with mouse, rat, cow, dog, guinea pig, horse, rabbit, and even yeast models

• Conjugation status: Primarily unconjugated antibodies that require secondary detection

How should I design Western blot experiments using KNSTRN antibodies?

For optimal Western blot detection of KNSTRN:

• Sample preparation:

  • Use appropriate lysis buffers (e.g., RIPA with protease inhibitors)

  • Load 20-40 μg of total protein per lane

• Gel electrophoresis and transfer:

  • Use 10-12% SDS-PAGE gels

  • Transfer to PVDF or nitrocellulose membranes

• Antibody incubation:

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

  • Primary antibody: Dilute KNSTRN antibody at 1:500-1:2000 as recommended

  • Secondary antibody: Use appropriate HRP-conjugated secondary (typically anti-rabbit)

• Detection considerations:

  • Expected molecular weight: ~34-35 kDa, though calculated weights of 26/31/35 kDa have been reported for different isoforms

  • Include appropriate positive controls (cell lines with known KNSTRN expression)

What are the key considerations for immunohistochemistry with KNSTRN antibodies?

When performing IHC with KNSTRN antibodies:

• Tissue preparation:

  • Formalin-fixed, paraffin-embedded (FFPE) sections (5 μm thickness)

  • Consider antigen retrieval methods (heat-induced epitope retrieval in citrate buffer pH 6.0)

• Staining protocol:

  • Blocking: 5-10% normal serum from secondary antibody host species

  • Primary antibody: Typical dilutions range from 1:200 to 1:500

  • Detection system: Use appropriate secondary antibody and visualization method (DAB, fluorescence)

• Controls:

  • Include positive controls (tissues known to express KNSTRN)

  • Include negative controls (primary antibody omission)

  • Consider dual staining with Ki67 to correlate with proliferative status

• Evaluation:

  • KNSTRN primarily shows nuclear and cytoplasmic localization

  • Quantify staining intensity and percentage of positive cells

How can I validate the specificity of a KNSTRN antibody?

Antibody validation is crucial for reliable results. Methods include:

• Genetic approaches:

  • KNSTRN knockdown/knockout: Compare antibody signal in wild-type vs. KNSTRN-depleted samples

  • KNSTRN overexpression: Verify increased signal in cells overexpressing KNSTRN

• Technical approaches:

  • Western blot: Confirm single band at expected molecular weight (~35 kDa)

  • Peptide competition: Pre-incubate antibody with immunizing peptide to block specific binding

  • Cross-validation: Compare results from antibodies targeting different KNSTRN epitopes

  • Compare reactivity across multiple cell lines with varying KNSTRN expression levels

• Functional correlation:

  • Verify subcellular localization matches expected pattern during mitosis

  • Correlate with known KNSTRN binding partners (e.g., SPAG5)

What could explain discrepancies between predicted and observed molecular weights of KNSTRN?

Several factors may contribute to molecular weight discrepancies:

• Post-translational modifications:

  • Phosphorylation during mitosis may alter migration

  • Other modifications could impact apparent molecular weight

• Protein isoforms:

  • Up to 3 different isoforms have been reported (26, 31, and 35 kDa)

  • Alternative splicing can produce variants of different sizes

• Technical factors:

  • Gel percentage and running conditions affecting migration

  • Buffer systems and sample preparation methods

  • Protein standards calibration

• Antibody specificity:

  • Different antibodies may recognize different isoforms or modified forms

  • Cross-reactivity with related proteins

To resolve discrepancies, researchers should compare results using antibodies targeting different epitopes and consider isoform-specific PCR to determine which variants are expressed in their experimental system.

How can I troubleshoot weak or absent KNSTRN signal in Western blots?

When facing weak or absent KNSTRN signals:

• Sample-related factors:

  • Verify KNSTRN expression in your sample (check literature/databases)

  • Increase protein loading (up to 50-60 μg)

  • Check protein extraction method (consider different lysis buffers)

  • Use fresh samples or add additional protease inhibitors

• Antibody-related factors:

  • Increase antibody concentration (try 1:500 or more concentrated)

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

  • Try different KNSTRN antibodies targeting other epitopes

  • Check antibody storage conditions and expiration

• Detection-related factors:

  • Use more sensitive detection methods (ECL Plus, fluorescent secondary antibodies)

  • Increase exposure time

  • Try signal enhancement systems

• Technical optimizations:

  • Optimize blocking conditions (try BSA instead of milk)

  • Increase washing stringency to reduce background

  • Use fresh transfer buffer and ensure complete transfer

How should I interpret KNSTRN expression patterns in cancer versus normal tissues?

When analyzing KNSTRN expression patterns:

How can KNSTRN antibodies be utilized to study mitotic spindle dynamics?

For investigating mitotic spindle dynamics:

• Immunofluorescence microscopy approaches:

  • Co-stain with KNSTRN antibody and microtubule markers (α-tubulin)

  • Use cell cycle phase-specific markers (pH3, cyclin B1)

  • Employ super-resolution microscopy for detailed localization

  • Consider live-cell imaging with fluorescently tagged KNSTRN

• Biochemical approaches:

  • Immunoprecipitate KNSTRN to identify interaction partners during different mitotic phases

  • Analyze phosphorylation status during mitotic progression

  • Study kinetochore association dynamics using chromatin immunoprecipitation

• Functional studies:

  • Combine with KNSTRN knockdown/overexpression to assess effects on spindle stability

  • Evaluate microtubule-kinetochore attachments after KNSTRN perturbation

  • Analyze chromosome segregation errors in KNSTRN-depleted cells

• Quantitative assessments:

  • Measure kinetochore oscillations and dynamics of microtubule plus-ends

  • Quantify metaphase-to-anaphase transition timing

  • Assess spindle pole architecture maintenance

What approaches can be used to study KNSTRN involvement in chromosomal instability and cancer?

To investigate KNSTRN's role in chromosomal instability:

• Cytogenetic analyses:

  • Karyotyping to detect gross chromosomal abnormalities

  • Fluorescence in situ hybridization (FISH) to identify specific chromosomal translocations

  • Multiplex-FISH for comprehensive chromosomal analysis

• Cell division assessments:

  • Time-lapse microscopy to monitor mitotic progression

  • Quantification of mitotic errors (lagging chromosomes, multipolar spindles)

  • Analysis of micronuclei formation as indicator of chromosomal instability

• Genetic approaches:

  • CRISPR/Cas9-mediated knockout of KNSTRN

  • Introduction of cancer-associated KNSTRN mutations

  • Rescue experiments with wild-type vs. mutant KNSTRN

• Clinical correlations:

  • Analyze KNSTRN mutation status in cancer samples

  • Correlate expression with chromosomal instability markers

  • Assess relationship with tumor mutation burden (TMB)

How can KNSTRN expression be correlated with immune infiltration in cancer research?

For studying KNSTRN-immune infiltration relationships:

• Bioinformatic approaches:

  • Use tools like TIMER2 to evaluate associations between KNSTRN expression and immune cell infiltration

  • Analyze correlation with T regulatory cells, Tgd, and natural killer cells

  • Calculate immune scores, stromal scores, and tumor purity using ESTIMATE package

• Multi-parameter immune profiling:

  • Multiplex immunohistochemistry for KNSTRN and immune markers

  • Flow cytometry analysis of tumor-infiltrating lymphocytes

  • Spatial transcriptomics to map KNSTRN expression relative to immune niches

• Functional validation:

  • In vitro co-culture of cancer cells and immune cells with KNSTRN modulation

  • Analysis of cytokine production and immune activation markers

  • Assessment of tumor cell susceptibility to immune-mediated killing

• Clinical integration:

  • Correlate KNSTRN expression with response to immunotherapy

  • Analyze relationship with immunosuppressive mechanisms

  • Stratify patients based on combined KNSTRN and immune signature

How can KNSTRN antibodies be used as prognostic biomarkers in cancer?

For prognostic applications:

What is the relationship between KNSTRN mutations and cancer development?

The relationship between KNSTRN mutations and carcinogenesis:

• Mutation profiling:

  • KNSTRN is among the top three frequently mutated genes in cutaneous squamous cell carcinomas (SCCs)

  • Different mutation types (SNV, indel, CNV, rearrangement) occur across cancer types

  • "Deep deletion" CNV is frequent in mesothelioma (MESO) and uterine carcinosarcoma (UCS)

  • SNV mutations are common in skin cancer (SKCM)

• Functional consequences:

  • Mutations may disrupt chromosome segregation during mitosis

  • Can lead to chromosomal instability and aneuploidy

  • May affect interaction with binding partners (e.g., SPAG5)

• Clinical correlations:

  • Association with UV signature mutations in skin cancers

  • Potential relationship with tumor mutation burden (TMB)

  • Possible impact on treatment response

• Mechanistic studies:

  • Introduction of cancer-associated mutations in cell models

  • Analysis of mitotic defects in mutant cells

  • Evaluation of downstream signaling pathway alterations

How does KNSTRN interact with cell cycle regulation pathways in cancer?

KNSTRN's role in cell cycle regulation:

• Cell cycle protein interactions:

  • KNSTRN overexpression enhances expression of key cell cycle regulators including CDK4, CDK6, and cyclin D3

  • Promotes G1/S phase transition in breast cancer cells

  • Gene set enrichment analysis suggests KNSTRN regulates cell cycle progression

• Experimental approaches:

  • Cell synchronization and cell cycle analysis after KNSTRN modulation

  • EdU incorporation assays to measure proliferation

  • CCK-8 and colony formation assays to assess cell viability

  • Western blot analysis of cell cycle checkpoint proteins

• Signaling pathway integration:

  • KNSTRN promotes tumorigenesis through AKT activation in bladder cancers

  • Single-cell transcriptome data analysis can reveal KNSTRN-regulated pathways

  • Potential interaction with other oncogenic or tumor suppressor pathways

• Therapeutic implications:

  • Potential synergy with cell cycle inhibitors (CDK4/6 inhibitors)

  • Possible synthetic lethality approaches

  • Identification of vulnerable nodes in KNSTRN-overexpressing tumors

Table 1: KNSTRN expression patterns across 30 solid tumor types from TCGA database