KRT16 Monoclonal Antibody

What is KRT16 and what cellular roles does it play?

Keratin 16 (KRT16) is a type I cytokeratin protein that forms intermediate filaments through heterodimeric assembly with type II cytokeratins. It is part of the cytokeratin family (cytokeratins 9-23 for type I) that maintains structural integrity in epithelial cells. KRT16 plays critical roles in cellular differentiation and tissue specialization. Recent research has revealed that KRT16 functions extend beyond structural support to include roles in cell motility regulation and epithelial-to-mesenchymal transition (EMT). KRT16 has been implicated in regulating the production of innate danger signals and cytokine activation following epidermal barrier breach, suggesting its importance in wound healing and inflammatory responses . These diverse functions make KRT16 relevant to multiple research areas including cancer biology, dermatology, and immunology.

In which tissue types is KRT16 normally expressed?

KRT16 expression demonstrates a specific tissue distribution pattern that makes it valuable as a diagnostic marker. It is primarily expressed in:

• Benign stratified squamous epithelium

• Squamous cell carcinoma of the head and neck

• Luminal cells of mammary gland

• Sweat ducts

• Suprabasal keratinocytes (as indicated by its role as a suprabasal keratinocyte marker)

Importantly, KRT16 is notably absent in non-invasive breast carcinomas and normal breast tissue, making its presence potentially significant for cancer diagnostics . This distinct expression pattern allows researchers to use KRT16 as a marker for specific epithelial cell populations and to identify the origin of metastatic tumors.

What are the recommended applications for KRT16 monoclonal antibodies?

KRT16 monoclonal antibodies are versatile research tools applicable to multiple experimental approaches. Based on validated applications from antibody manufacturers and research publications, recommended applications include:

• Western blotting (WB): Typically using dilutions of 1:500-1:1,000 depending on the specific antibody

• Immunocytochemistry (ICC): For cellular localization studies

• Immunohistochemistry (IHC): For tissue section analysis

• Dot blotting (DB): Particularly useful for autoantibody detection studies

• Flow cytometry: For analyzing circulating tumor cells and other single-cell applications

When designing experiments, researchers should verify the specific applications validated for their particular KRT16 antibody clone, as performance may vary between manufacturers and clones. Preliminary titration experiments are recommended to determine optimal antibody concentrations for each application.

What storage and handling procedures should be followed for KRT16 monoclonal antibodies?

Proper storage and handling of KRT16 monoclonal antibodies is essential for maintaining reactivity and specificity. Follow these research-validated guidelines:

• For antibodies containing sodium azide preservative: Store at 2-8°C (refrigerated)

• For antibodies without preservatives: Store at -20°C to -80°C

• Avoid repeated freeze-thaw cycles that can degrade antibody performance

• Typical antibody formulations include:

  • Concentration: 200μg/ml (purified by Protein A/G)

  • Buffer: 10mM PBS with 0.05% BSA & 0.05% azide (standard formulation)

  • Alternative formulation: 1.0mg/ml without BSA & azide (for applications sensitive to these components)

When handled properly, KRT16 antibodies typically maintain stability for approximately 24 months. Always centrifuge briefly before opening vials to collect any solution that may have gathered in the cap during shipping or storage.

What controls are essential when using KRT16 monoclonal antibodies?

Implementing appropriate controls is crucial for experimental validity when using KRT16 monoclonal antibodies. Essential controls include:

• Positive tissue controls: Use tissues known to express KRT16, such as:

  • Stratified squamous epithelium samples

  • Squamous cell carcinoma of the head and neck

  • Sweat ducts or mammary gland luminal cells

• Negative tissue controls: Include tissues known to lack KRT16 expression:

  • Normal breast tissue

  • Non-invasive breast carcinomas

• Antibody controls:

  • Isotype control (Mouse IgG1, kappa) to assess non-specific binding

  • Secondary antibody-only control to evaluate background

• Knockdown validation: When possible, include KRT16 knockdown samples as specificity controls. The published siRNA sequences for KRT16 knockdown are:

  • KRT16siRNA1: GGAGAUGCUUGCUCUGAGA

  • KRT16siRNA2: GGCCAGAGCUCCUAGAACU

  • KRT16siRNA3: GGAACAAGAUCAUUGCGGC

  • KRT16siRNA4: GCGGAGAUGUGAACGUGGA

These controls ensure that signals detected are specific to KRT16 and not due to non-specific binding or technical artifacts.

How does KRT16 expression correlate with cancer progression and metastasis?

KRT16 has emerged as a significant marker in cancer progression and metastasis, particularly in breast cancer. In silico analysis has demonstrated a positive correlation between KRT16 gene expression and shorter relapse-free survival in large breast cancer patient datasets . This correlation indicates that KRT16 expression is associated with higher tumor aggressiveness.

Research findings demonstrate several key relationships between KRT16 and metastatic potential:

• High KRT16 protein expression is associated with an intermediate mesenchymal phenotype in cancer cells

• Functional studies show that KRT16 has regulatory effects on epithelial-to-mesenchymal transition (EMT)

• KRT16 overexpression significantly enhances cell motility (p < 0.001), suggesting direct involvement in metastatic processes

• In metastatic breast cancer patients, 64.7% of detected circulating tumor cells (CTCs) expressed KRT16

• KRT16 expression in CTCs was associated with shorter relapse-free survival (p = 0.0042)

These findings collectively suggest that KRT16 functions as a metastasis-associated protein that promotes EMT and positively regulates cellular motility. When designing studies to investigate KRT16's role in cancer progression, researchers should consider multiple techniques (RNA expression, protein abundance, and cellular localization) to comprehensively characterize its involvement in their specific cancer model.

What methods are recommended for studying KRT16's role in epithelial-to-mesenchymal transition (EMT)?

To investigate KRT16's role in EMT, researchers should employ a multifaceted approach combining molecular, cellular, and functional assays:

• Expression analysis:

  • RT-qPCR to quantify KRT16 mRNA levels (as performed in metastatic breast cancer studies)

  • Western blot for protein abundance assessment using recommended antibody dilutions (1:500)

  • Immunocytochemistry for subcellular localization

• EMT marker correlation:

  • Concurrent analysis of established EMT markers (E-cadherin, vimentin, N-cadherin)

  • Co-immunoprecipitation to identify potential KRT16 interaction partners during EMT

• Functional modification approaches:

  • Overexpression studies using transfected KRT16 expression vectors

  • RNA interference using validated siRNA sequences:

    • KRT16siRNA1: GGAGAUGCUUGCUCUGAGA

    • KRT16siRNA2: GGCCAGAGCUCCUAGAACU

    • KRT16siRNA3: GGAACAAGAUCAUUGCGGC

    • KRT16siRNA4: GCGGAGAUGUGAACGUGGA

• Functional assays:

  • Cell motility assays (demonstrated to be significantly affected by KRT16 expression, p < 0.001)

  • Invasion assays through extracellular matrix

  • Cell adhesion assessments

• In vivo models:

  • Mouse models examining KRT16 expression in primary tumors versus metastases

  • Analysis of circulating tumor cells for KRT16 expression

By integrating these methodologies, researchers can establish both correlative and causal relationships between KRT16 and EMT processes in their specific experimental models.

What protocols are recommended for detecting KRT16 in circulating tumor cells (CTCs)?

Detection of KRT16 in circulating tumor cells requires specialized protocols to ensure sensitivity and specificity. Based on successful research methodologies, the following approach is recommended:

CTC Isolation and KRT16 Detection Protocol:

• CTC Enrichment:

  • Use density gradient centrifugation or specialized CTC isolation platforms

  • Alternative: Immunomagnetic separation using epithelial markers (EpCAM)

• Immunocytochemical Detection:

  • Fix isolated cells with 4% paraformaldehyde (10 minutes at room temperature)

  • Permeabilize with 0.1% Triton X-100 in PBS (5 minutes)

  • Block with 3% BSA in PBS (60 minutes)

  • Incubate with anti-KRT16 primary antibody (recommended dilution 1:500)

  • Detect using fluorescently-labeled secondary antibodies (e.g., IrDye 800CW Goat anti-mouse IgG at 1:20,000)

• Validation Controls:

  • Include breast cancer cell lines with known KRT16 expression as positive controls

  • Use non-epithelial cells (e.g., leukocytes) as negative controls

  • Employ multiple markers for CTC identification to distinguish true CTCs from other circulating cells

• Analysis Considerations:

  • Quantify percentage of KRT16-positive CTCs (reported as 64.7% in metastatic breast cancer patients)

  • Correlate KRT16 expression with clinical outcomes (relapse-free survival)

  • Consider multi-parameter analysis including other EMT markers

This protocol has successfully identified KRT16-expressing CTCs that were associated with shorter relapse-free survival (p = 0.0042) in metastatic breast cancer patients, demonstrating its clinical relevance .

How can I validate KRT16 antibody specificity in my experimental model?

Rigorous validation of KRT16 antibody specificity is critical for experimental reliability. Implement these comprehensive validation strategies:

• Genetic Modification Controls:

  • Use non-targeting pool as negative control (e.g., ON-TARGETplus Non-targeting Pool)

  • Assess knockdown efficiency by RT-qPCR and western blot 48 hours post-transfection

• Technical Validation:

  • Perform western blotting with recombinant KRT16 protein

  • Compare multiple KRT16 antibody clones when possible

  • Conduct peptide competition assays to confirm epitope specificity

• Cross-reactivity Assessment:

  • Test antibody against other keratin family members, particularly those with sequence homology

  • Include tissues with known expression patterns of various keratins

• Multi-technique Concordance:

  • Confirm antibody performance across multiple platforms (western blot, immunocytochemistry, flow cytometry)

  • Assess correlation between protein detection and mRNA expression (RT-qPCR)

• Quantitative Validation:

  • Generate standard curves using recombinant KRT16 protein

  • Determine limits of detection and quantification

  • Assess linear range of antibody performance

This comprehensive validation approach ensures that experimental observations are specifically attributable to KRT16 rather than technical artifacts or cross-reactivity with related proteins.

What is the evidence for KRT16 as an autoantigen in complex regional pain syndrome (CRPS)?

Evidence for KRT16 as an autoantigen in CRPS comes from both mouse models and human patient samples. Using a tibia fracture/cast immobilization CRPS model, researchers identified KRT16 as a primary autoantigen target through several lines of evidence:

• Increased Expression in CRPS-affected Tissue:

  • KRT16 mRNA levels were significantly elevated in mouse skin 3 weeks following fracture

  • Protein levels were similarly increased in affected tissues

• Autoantibody Detection Methodology:

  • Dot blot analysis using recombinant KRT16 protein demonstrated:

    • Increased binding of sera from fracture mice to KRT16

    • Increased binding of sera from CRPS patients to KRT16

  • Similar experiments with other candidate proteins (PRPH) showed no differential binding

• Clinical Correlation:

  • No correlation was observed between KRT16 immunoreactivity and CRPS duration in patients, suggesting it may be present throughout the disease course

• Experimental Controls:

  • Experiments employed appropriate controls including non-injured mice and non-CRPS human sera

  • Technical validation included quantitative dot blot analysis protocols

• Biological Context:

  • KRT16 is instrumental in regulating innate danger signals and cytokine activation after skin barrier breach

  • This parallels the trophic changes that often accompany CRPS

This evidence supports KRT16 as a potential biomarker for CRPS, suggesting an autoimmune etiology for this challenging condition. For researchers investigating this connection, the methodologies outlined in the original study (including dot blot analysis with recombinant KRT16) provide a validated approach for detecting anti-KRT16 autoantibodies.

What technical considerations are important for multiplexed immunofluorescence with KRT16 antibodies?

Multiplexed immunofluorescence involving KRT16 antibodies requires careful technical optimization to ensure specificity, sensitivity, and compatibility with other markers. Key considerations include:

• Fluorophore Selection and Spectral Overlap:

  • When selecting fluorophores for KRT16 detection, consider these validated options:

    • CF®405S (Ex/Em: 404/431nm) - Note: Not recommended for low abundance targets due to higher background

    • CF®488A (Ex/Em: 490/515nm) - Good option for standard FITC/GFP channels

    • CF®568 (Ex/Em: 562/583nm) - Compatible with RFP/TRITC channels

    • CF®594 (Ex/Em: 593/614nm) - Red channel option with minimal overlap

  • Account for spectral overlap when designing panels with multiple markers

• Antibody Validation for Multiplexing:

  • Validate each antibody individually before multiplexing

  • Test for cross-reactivity between primary and secondary antibodies

  • Verify epitope accessibility in multiplexed staining conditions

• Protocol Optimization:

  • Sequential staining may be required for optimal results:

    1. Apply first primary antibody (e.g., KRT16)

    2. Detect with fluorescent secondary

    3. Block remaining free binding sites

    4. Apply subsequent antibodies sequentially

  • Alternative: Directly conjugated primary antibodies to eliminate cross-reactivity

  • Consider tyramide signal amplification for low-abundance targets

• Tissue-Specific Considerations:

  • Epitope retrieval methods may affect keratin detection differently than other targets

  • Autofluorescence in epithelial tissues may require specific quenching protocols

  • Keratin network density may impact antibody penetration

• Controls for Multiplexed Experiments:

  • Single-stained controls for each marker

  • Fluorescence-minus-one (FMO) controls

  • Absorption controls using recombinant KRT16 protein

Following these technical considerations will help ensure reliable multiplexed detection of KRT16 alongside other biomarkers of interest.

How does KRT16 function differ from other keratin family members in pathological conditions?

KRT16 exhibits distinct functional properties and expression patterns compared to other keratin family members in various pathological conditions. Understanding these differences is crucial for accurate interpretation of experimental results:

• Unique Expression in Disease States:

  • Unlike many keratins, KRT16 is notably absent in normal breast tissue but becomes expressed in invasive breast carcinomas

  • KRT16 shows specific upregulation in suprabasal keratinocytes during wound healing and inflammatory skin conditions

  • It serves as a regional autoimmunity marker in conditions like alopecia areata and CRPS

• Functional Distinctions in Cancer Progression:

  • KRT16 specifically promotes cell motility, unlike many structural keratins

  • It demonstrates a regulatory effect on EMT, whereas many keratins are downregulated during EMT

  • KRT16 expression in circulating tumor cells correlates with shorter relapse-free survival (p = 0.0042)

• Regulatory Network Interactions:

  • KRT16 uniquely regulates the production of innate danger signals following epidermal barrier breach

  • It influences cytokine activation and regulators of skin barrier function

  • Unlike purely structural keratins, KRT16 appears to have signaling functions relevant to inflammatory processes

• Heterodimeric Partners:

  • As a type I keratin, KRT16 forms specific heterodimers with type II keratins (keratins 1-8)

  • These specific pairing relationships influence tissue localization and function

  • Understanding these pairing relationships is essential when interpreting co-expression studies

• Methodological Implications:

  • KRT16 antibodies may require different detection conditions than antibodies against other keratins

  • Validation strategies should account for the specific expression patterns of KRT16 versus other family members

These distinctions highlight why KRT16-specific research requires targeted approaches rather than generalizing findings from studies of other keratin family members.

What are the recommended troubleshooting steps for inconsistent KRT16 immunoblotting results?

When encountering inconsistent results in KRT16 immunoblotting experiments, follow this systematic troubleshooting approach:

• Sample Preparation Issues:

  • Ensure complete protein extraction: Keratins are insoluble intermediate filament proteins requiring appropriate lysis buffers (consider SDS or urea-based buffers)

  • Prevent protein degradation: Add fresh protease inhibitors to all buffers

  • Verify protein concentration: Reconfirm BCA or Bradford assay results

  • Check heat denaturation: KRT16 requires complete denaturation (95°C for 5 minutes)

• Gel Electrophoresis Optimization:

  • Verify migration pattern: KRT16 has a molecular weight of approximately 48 kDa

  • Use appropriate gel percentage: 10-12% acrylamide gels typically work well

  • Include positive control: Recombinant KRT16 or lysate from cells known to express KRT16

  • Consider gradient gels for better resolution of keratin family members

• Antibody-Specific Issues:

  • Titrate antibody concentration: Test dilution ranges from 1:500 to 1:5,000

  • Verify antibody storage conditions: Avoid repeated freeze-thaw cycles

  • Check lot-to-lot variation: Request technical support if new lot produces different results

  • Consider alternative KRT16 antibody clones if persistent issues occur

• Detection System Troubleshooting:

  • For fluorescent detection: Use validated secondary antibodies like IrDye 800CW Goat anti-rabbit IgG (1:20,000)

  • For chemiluminescent detection: Optimize substrate exposure time

  • Background issues: Increase blocking time and washing steps

  • Signal strength: Consider signal amplification methods for low-abundance samples

• Experimental Controls to Implement:

  • Positive control: MDA-MB-468 cells are documented to express KRT16

  • Negative control: Cells with KRT16 knockdown using validated siRNA sequences

  • Loading control: GAPDH (using anti-GAPDH at 1:5,000 dilution)

By systematically addressing these potential issues, researchers can identify and resolve the specific factors causing inconsistent KRT16 immunoblotting results.

Validated Antibody Applications and Dilutions

The following table summarizes recommended applications and dilutions for KRT16 antibody use in various experimental protocols:

KRT16 siRNA Sequences for Knockdown Validation

For KRT16 knockdown experiments, the following validated siRNA sequences have been successfully used:

KRT16 Expression in Tissue Types

This table summarizes the tissue expression pattern of KRT16 for reference when selecting appropriate experimental controls: