KRT17 Monoclonal Antibody

What is KRT17 and what is its biological significance?

Cytokeratin 17 (KRT17) is a type I intermediate filament protein normally expressed in the basal cells of complex epithelia but not in stratified or simple epithelia. It serves as an excellent marker to distinguish myoepithelial cells from luminal epithelium in various glands including mammary, sweat, and salivary glands . KRT17 is also expressed in various epithelial cell types, such as bronchial epithelial cells and skin appendages . From a developmental perspective, KRT17 is often considered an epithelial stem cell marker because KRT17 antibodies mark basal cell differentiation . This protein plays a significant role in cell structural integrity and signaling pathways that influence cell growth, making it an important target for both basic research and disease studies.

What are the primary applications of KRT17 monoclonal antibodies?

KRT17 monoclonal antibodies have diverse applications across multiple experimental platforms. Based on currently available products, the primary applications include:

• Western Blotting (WB): Used for protein detection with recommended dilutions ranging from 1:200 to 1:2000 .

• Immunohistochemistry (IHC): Applied to both paraffin-embedded and frozen tissue sections with optimal dilutions typically between 1:50 to 1:200 .

• Flow Cytometry (FACS): Employed for cell sorting and quantitative analysis at dilutions of 1:10 to 1:1000 .

• Immunocytochemistry (ICC)/Immunofluorescence (IF): Used for cellular localization studies with recommended dilutions from 1:10 to 1:2000 .

These applications make KRT17 antibodies versatile tools for researchers investigating epithelial biology, developmental processes, and various pathological conditions. For all applications, it's important to note that the optimal working dilution should be determined experimentally by the end user based on their specific sample types and conditions .

How do I select the appropriate KRT17 antibody clone for my research?

Selecting the appropriate KRT17 antibody clone depends on your experimental design, species of interest, and target application. Consider these factors:

• Target Species Reactivity: Various KRT17 antibodies show different cross-reactivity profiles. For example, clone E3 is reactive with human and rat samples , while other clones may have broader reactivity including mouse, cow, and pig models .

• Antibody Type: Both monoclonal and polyclonal options are available. Monoclonal antibodies like clone E3 offer high specificity and reproducibility, while polyclonal antibodies may provide higher sensitivity but potentially more background .

• Target Epitope: Some antibodies target specific regions of KRT17, such as C-terminal regions or particular amino acid sequences (e.g., AA 133-432, AA 252-393). Select based on your region of interest or to avoid potential cross-reactivity with other cytokeratins .

• Application Compatibility: Verify that your selected antibody has been validated for your intended application. For instance, the E3 clone has been validated for WB, IHC, FACS, and ICC applications .

• Conjugation Needs: Determine whether you need an unconjugated antibody or one conjugated with fluorescent dyes (like CF® dyes) for direct detection methods .

For comprehensive studies, antibodies like the E3 clone, which was derived from immunization with cytoskeletal preparations from rat colon, offer versatility across multiple applications .

How does KRT17 expression correlate with cancer subtypes and clinical outcomes?

KRT17 expression shows significant correlations with various cancer subtypes and can predict clinical outcomes, making it valuable in translational research:

• Small Cell Lung Cancer (SCLC): KRT17 is expressed at significantly higher levels in SCLC compared to lung adenocarcinoma (LADC), making it a potential diagnostic marker for differentiating these cancer subtypes .

• Triple-Negative Breast Carcinoma: Approximately 85% of triple-negative breast carcinomas show immunoreactivity with basal cytokeratins including KRT17. Importantly, cases of triple-negative breast carcinoma with KRT17 expression demonstrate more aggressive clinical courses, suggesting its value as a prognostic marker .

• Ampullary Cancer Differentiation: KRT17 plays a crucial role in the histologic differentiation of ampullary cancer subtypes. KRT17 and MUC1 immunoreactivity typically represents the pancreatobiliary subtype, whereas MUC2 and CDX-2 positivity defines the intestinal subtype. This distinction is crucial for treatment planning and prognosis assessment .

These correlations highlight the importance of precise KRT17 detection in cancer pathology and suggest its potential as both a diagnostic and prognostic biomarker in clinical settings.

What are the best practices for quantifying KRT17 expression in tissue samples?

Quantifying KRT17 expression in tissue samples requires standardized approaches for reproducible results:

• Antibody Selection and Validation: Use well-characterized antibodies with confirmed specificity. The E3 clone has been extensively validated for IHC applications in human tissues .

• Optimal Staining Protocol: For paraffin-embedded sections, a 1:50 to 1:200 dilution of KRT17 monoclonal antibody is typically recommended with peroxidase-conjugate and DAB chromogen for visualization . The protocol should include appropriate antigen retrieval methods, typically heat-induced epitope retrieval in citrate buffer (pH 6.0).

• Positive and Negative Controls: Include known positive tissues (e.g., skin samples where KRT17 is expressed in hair follicles) and negative controls (primary antibody omission) in each staining batch .

• Scoring Systems: Implement standardized scoring systems based on:

  • Staining intensity (0, 1+, 2+, 3+)

  • Percentage of positive cells (0-100%)

  • H-score calculation (intensity × percentage, range 0-300)

• Digital Image Analysis: Employ digital pathology platforms with validated algorithms for unbiased quantification of staining intensity and distribution patterns.

• Normalization Strategy: Normalize expression against housekeeping proteins when performing Western blot quantification to account for loading variations.

These approaches ensure reliable quantification of KRT17 expression for comparative studies across different tissue samples or experimental conditions.

What are the considerations for multiplexing KRT17 antibodies with other markers?

Multiplexing KRT17 antibodies with other markers requires careful planning to avoid technical pitfalls and ensure valid results:

• Antibody Species and Isotype: When multiplexing, select primary antibodies from different host species or different isotypes if from the same species. For example, if using the mouse monoclonal E3 clone (IgG2b, kappa) , pair it with antibodies from rabbit or goat, or with mouse antibodies of different isotypes (IgG1, IgM).

• Spectral Compatibility: For fluorescent multiplexing, choose fluorophores with minimal spectral overlap. Primary antibodies are available with various fluorescent CF® dyes, though note that blue fluorescent dyes like CF®405S and CF®405M are not recommended for low-abundance targets due to higher non-specific background .

• Sequential Staining Consideration: For challenging combinations, consider sequential staining approaches with intermittent blocking steps to minimize cross-reactivity.

• Epitope Accessibility: Ensure that staining conditions (antigen retrieval method, buffers) are compatible for all antibodies in the panel.

• Co-localization Analysis: When studying co-expression patterns, implement appropriate co-localization algorithms and controls to validate findings.

• Common Marker Combinations: In epithelial research, KRT17 is frequently multiplexed with:

  • Other cytokeratin markers (CK5/6, CK7, CK20) for epithelial subtyping

  • MUC1, MUC2, and CDX-2 for gastrointestinal and pancreatobiliary differentiation

  • Proliferation markers (Ki-67) to assess growth dynamics in KRT17-positive cells

These considerations ensure valid interpretation of multiplex staining results and maximize the information obtained from limited tissue samples.

How can I optimize KRT17 antibody protocols for challenging sample types?

Optimizing KRT17 antibody protocols for challenging samples requires systematic troubleshooting approaches:

• Formalin-Fixed, Paraffin-Embedded (FFPE) Tissues with High Autofluorescence:

  • For immunohistochemistry: Use the recommended 1:200 dilution with peroxidase-conjugate and DAB chromogen

  • Incorporate autofluorescence quenching steps (e.g., Sudan Black B treatment)

  • Consider tyramide signal amplification for low-expression samples

• Poorly Fixed or Degraded Samples:

  • Increase antibody concentration slightly (e.g., 1:50 instead of 1:100)

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

  • Enhance antigen retrieval by optimizing pH and duration

• High-Background Clinical Samples:

  • Implement additional blocking steps with serum matching the host of the secondary antibody

  • Include protein blocking agents (BSA, casein) at higher concentrations

  • Consider using polymer-based detection systems instead of biotin-based methods

• Samples with Low KRT17 Expression:

  • Avoid blue fluorescent dyes (CF®405S, CF®405M) which have lower fluorescence and higher background

  • Utilize amplification systems such as HRP-polymer conjugates

  • Consider longer exposure times or more sensitive detection methods

• Long-Term Stored Samples:

  • Freshly cut sections from stored blocks may require more aggressive antigen retrieval

  • Adjust antibody concentration based on sample age and storage conditions

By systematically optimizing these parameters, researchers can achieve reliable KRT17 detection even in challenging sample types that might otherwise yield inconclusive results.

What are the optimal storage conditions for KRT17 monoclonal antibodies?

Proper storage of KRT17 monoclonal antibodies is crucial for maintaining their performance over time. Based on manufacturer recommendations:

• Short-term Storage: Store at 4°C when the antibody will be used within a few weeks .

• Long-term Storage: For extended periods, store at -20°C to preserve antibody activity .

• Aliquoting: To prevent repeated freeze-thaw cycles, which can deteriorate antibody quality, divide the stock solution into small aliquots before freezing .

• Buffer Composition: KRT17 antibodies are typically stored in PBS containing 0.05% BSA and 0.05% sodium azide as preservatives . Note that sodium azide is a hazardous substance that should be handled by trained staff only .

• Shipping and Temporary Storage: Antibodies can typically withstand shipping at ambient temperatures for several days, but should be transferred to recommended storage conditions immediately upon receipt.

• Stability Considerations: Most antibody conjugates have different stability profiles. For example, CF® dye and biotin conjugates typically have lead times of up to one week, while fluorescent protein and enzyme conjugates may require 2-3 weeks .

Following these storage recommendations ensures optimal antibody performance and extends the usable life of these valuable research reagents.