KIF11 Monoclonal Antibody

What is KIF11 and why is it important in cancer research?

The importance of KIF11 extends beyond its prognostic value. Studies have demonstrated that KIF11 enhances the self-renewal ability of breast cancer cells by participating in the Wnt/β-catenin pathway, thereby promoting cancer stem cell characteristics . This multifaceted role in cancer progression makes KIF11 a valuable target for both diagnostic and therapeutic applications.

What are the standard methods for detecting KIF11 expression using monoclonal antibodies?

Several methods can effectively detect KIF11 expression using monoclonal antibodies:

• Immunohistochemistry (IHC): This is the most widely used technique for evaluating KIF11 protein expression in clinical samples. IHC allows visualization of KIF11 expression patterns within tissue architecture and cellular compartments. In studies of endometroid cancer and gastric cancer, researchers have successfully used IHC to correlate KIF11 expression with clinicopathological features and patient outcomes .

• Western Blotting: For quantitative analysis of KIF11 protein levels, western blotting provides reliable results. This technique has been applied to confirm KIF11 expression in cell lines and to validate knockdown efficiency in RNA interference experiments .

• Immunofluorescence: This method enables precise localization of KIF11 within cellular compartments and has been particularly useful in studying its colocalization with other proteins such as ZBP1 .

• Flow Cytometry: For analyzing KIF11 expression in heterogeneous cell populations, flow cytometry offers high-throughput capabilities and can be combined with other markers to identify specific cell subpopulations .

What controls should be included when using KIF11 monoclonal antibodies?

Appropriate controls are essential for ensuring reliable results with KIF11 monoclonal antibodies:

• Positive Controls: Include tissues or cell lines known to express high levels of KIF11, such as breast cancer cell lines (MCF-7, SKBR-3) or endometrial cancer tissues .

• Negative Controls: Use tissues with minimal KIF11 expression or include primary antibody omission controls.

• Isotype Controls: Include matched isotype controls to assess non-specific binding.

• Knockdown Validation: When possible, include KIF11 knockdown samples (using siRNA or shRNA) to confirm antibody specificity. Studies have shown that KIF11 can be effectively downregulated using targeted shRNAs, reducing expression to approximately 40% of control levels .

• Cross-Validation: Verify KIF11 expression using multiple detection methods (e.g., both IHC and western blot) to enhance confidence in your findings.

How can KIF11 monoclonal antibodies be used to study the relationship between KIF11 and cancer stem cells?

KIF11 has been implicated in cancer stem cell (CSC) biology, particularly in breast cancer . Researchers can employ KIF11 monoclonal antibodies to investigate this relationship through several approaches:

• Side Population (SP) Analysis: Flow cytometry using KIF11 antibodies in combination with CSC markers can help quantify the proportion of side population cells with KIF11 expression. Research has shown that silencing KIF11 significantly reduces the proportion of SP cells in breast cancer cell lines .

• Mammosphere Formation Assays: These assays assess the self-renewal capacity of cancer cells. Following treatments targeting KIF11 (inhibitors or siRNA), researchers can use KIF11 antibodies to confirm knockdown efficiency and correlate it with changes in mammosphere size and number. Studies have demonstrated that silencing KIF11 reduces both the size and number of mammospheres formed by breast cancer cells in vitro .

• Co-immunoprecipitation (Co-IP): KIF11 antibodies can be used in Co-IP experiments to identify interactions between KIF11 and components of stemness-related pathways such as Wnt/β-catenin. This approach helps elucidate the molecular mechanisms by which KIF11 influences cancer stem cell properties .

• In vivo Tumor Formation: Following manipulation of KIF11 expression, antibodies can be used to verify KIF11 status in xenograft tumor sections, correlating expression levels with tumor-initiating capacity .

What methodologies are recommended for studying KIF11's interaction with ZBP1 in mRNA transport?

KIF11 has been shown to interact with ZBP1 in regulating the transport of β-actin mRNA . To study this interaction:

• Co-immunoprecipitation (Co-IP): Use KIF11 monoclonal antibodies to pull down protein complexes and detect ZBP1 by immunoblotting. Research has demonstrated that both ZBP1 and KIF11 can be co-precipitated with β-actin-MS2 mRNA .

• Domain Mapping: By using antibodies against different domains of KIF11, researchers can identify which regions interact with ZBP1. Studies have shown that the tail domain of KIF11 (residues 762-1056) is sufficient and necessary for interaction with ZBP1 .

• Fluorescence In Situ Hybridization (FISH): Combine FISH for β-actin mRNA with immunofluorescence using KIF11 antibodies to visualize colocalization. This approach has revealed that inhibition of KIF11 activity or downregulation of KIF11 expression impairs β-actin mRNA localization .

• Protein Purification and Direct Binding Assays: Use recombinant KIF11 fragments and ZBP1 domains with KIF11 antibodies for detection in binding assays. Research has shown that the tail fragment of KIF11 efficiently binds to immobilized ZBP1-RRM12 domain .

How can researchers use KIF11 antibodies to evaluate its potential as a therapeutic target?

To assess KIF11's potential as a therapeutic target, researchers can employ several strategies:

What are the optimal protocols for using KIF11 monoclonal antibodies in immunohistochemistry?

For optimal IHC results with KIF11 monoclonal antibodies:

• Tissue Preparation:

  • Use formalin-fixed, paraffin-embedded (FFPE) tissues sectioned at 4-5 μm thickness

  • Include both tumoral and non-tumoral tissue sections when available for comparative analysis

• Antigen Retrieval:

  • Heat-induced epitope retrieval in citrate buffer (pH 6.0) or EDTA buffer (pH 9.0)

  • Microwave heating for 15-20 minutes has shown good results in multiple studies

• Antibody Incubation:

  • Optimization of antibody dilution is crucial (typically 1:100 to 1:500)

  • Incubation at 4°C overnight often yields better results than shorter incubations at room temperature

• Detection Systems:

  • HRP-conjugated secondary antibodies with DAB (3,3'-diaminobenzidine) substrate provide good contrast

  • For multiplex staining (e.g., KIF11 with KIF14), consider using fluorescent secondaries with distinct fluorophores

• Scoring System:

  • Implement a standardized scoring system that accounts for both staining intensity and percentage of positive cells

  • A commonly used approach is the H-score (0-300), calculated as: 1 × (% of cells with weak staining) + 2 × (% of cells with moderate staining) + 3 × (% of cells with strong staining)

How should researchers design KIF11 knockdown experiments with appropriate antibody validation?

When designing KIF11 knockdown experiments:

• Selection of RNAi Tools:

  • Use validated siRNA or shRNA sequences targeting different regions of KIF11 mRNA

  • Include at least two different targeting sequences to control for off-target effects

• Knockdown Validation:

  • Western blotting with KIF11 monoclonal antibodies provides quantitative assessment of knockdown efficiency

  • Aim for at least 50-60% reduction in protein expression; studies have achieved 56-59% reduction using specific shRNAs

• Functional Assays:

  • Include appropriate functional readouts based on research questions:

    • Cell proliferation/viability assays

    • Sphere formation assays for cancer stem cell properties

    • mRNA localization assays for transport functions

    • Invasion and migration assays

• Controls:

  • Non-targeting siRNA/shRNA controls

  • Parental cell lines without any treatment

  • Rescue experiments with siRNA-resistant KIF11 constructs to confirm specificity

What considerations are important when using KIF11 antibodies for studying combined biomarker expression?

When investigating KIF11 in combination with other biomarkers:

• Antibody Compatibility:

  • Ensure antibodies used in multiplex assays are raised in different host species to avoid cross-reactivity

  • Validate the specificity of each antibody individually before combined use

• Sequential Staining Protocols:

  • For co-expression studies (e.g., KIF11 and KIF14), sequential staining protocols may be necessary

  • Between rounds of staining, include thorough washing steps and consider using antibody stripping buffers if required

• Quantification Methods:

  • Develop consistent scoring methods for evaluating combined expression patterns

  • Consider creating expression profiles (e.g., KIF11highKIF14low) based on predetermined cutoffs

• Statistical Approaches:

  • Use appropriate statistical methods to analyze the prognostic value of combined biomarkers

  • Multivariate Cox regression analyses can help determine whether combined expression profiles provide independent prognostic information

What statistical methods should be used to analyze KIF11 expression data in relation to patient outcomes?

For robust statistical analysis:

How should researchers interpret varying KIF11 expression patterns across different cancer types?

When interpreting KIF11 expression across cancer types:

• Cross-Cancer Comparison Table:

• Tissue-Specific Considerations:

  • Consider normal expression patterns in corresponding non-malignant tissues

  • Evaluate KIF11 in the context of tissue-specific molecular subtypes

  • For gastric cancer, KIF11 expression is more frequent in intestinal phenotype tumors

• Functional Context:

  • Consider the role of KIF11 in different cellular processes specific to each cancer type

  • In breast cancer, focus on stem cell properties and Wnt/β-catenin pathway activation

  • In other contexts, consider mRNA transport functions or mitotic roles

What approaches can be used to study the relationship between KIF11 expression and treatment response?

To investigate KIF11 as a predictor of treatment response:

• Pre- and Post-Treatment Analysis:

  • Collect paired samples before and after therapy

  • Use KIF11 antibodies to compare expression levels and cellular localization

  • Correlate changes with clinical response metrics

• Cell Line Models:

  • Establish cell lines with varying KIF11 expression levels

  • Perform drug sensitivity assays and correlate with KIF11 status

  • Use KIF11 antibodies to confirm expression levels before and after treatment

• Pathway Analysis:

  • Investigate how KIF11 levels affect downstream signaling pathways relevant to therapy resistance

  • Studies have shown KIF11 involvement in the Wnt/β-catenin pathway in breast cancer cells

  • Levels of phosphorylated Erk1/2 were found to be affected by KIF11 knockdown in gastric cancer cells

• Combinatorial Approaches:

  • Test combinations of KIF11 inhibitors with standard therapies

  • Use KIF11 antibodies to monitor protein levels during combination treatment

  • Correlate expression patterns with synergistic effects

How can KIF11 monoclonal antibodies be utilized in studying lymphatic dysfunction in Microcephaly-Lymphedema syndrome?

Recent research has identified KIF11 mutations in Microcephaly-Lymphedema-Chorioretinopathy (MLC) syndrome . To investigate the role of KIF11 in lymphatic function:

• Patient-Derived Cell Models:

  • Use KIF11 antibodies to evaluate protein expression in lymphoblastoid cells from MLC patients

  • Research has shown that patients carrying pathogenic stop-gain variants in KIF11 have reduced mRNA and protein levels

• Co-localization Studies:

  • Employ KIF11 antibodies alongside lymphatic markers such as VEGFR3, Podoplanin, and PROX1

  • Studies have revealed that KIF11 is expressed in early human and mouse development with these lymphatic markers

• Functional Assays:

  • Use antibodies to verify KIF11 status in lymphatic endothelial cells (LECs) treated with EG5 inhibitors

  • Correlate KIF11 expression with changes in lymphatic function

What are the considerations for using KIF11 antibodies in high-throughput screening applications?

For high-throughput applications:

• Automation Compatibility:

  • Select KIF11 antibodies validated for automated immunostaining platforms

  • Optimize protocols for consistent staining across large sample sets

• Multiplexing Capabilities:

  • Evaluate antibodies for compatibility with multiplex immunofluorescence or mass cytometry

  • Consider conjugated KIF11 antibodies for direct detection without secondary antibodies

• Image Analysis:

  • Develop algorithms for automated quantification of KIF11 staining

  • Implement machine learning approaches for pattern recognition in complex tissues

• Quality Control:

  • Include tissue microarrays with known KIF11 expression patterns as controls

  • Implement batch correction methods to account for day-to-day variation