KRT17 Antibody

What is KRT17 and why is it significant in cancer research?

KRT17 (Keratin 17) is a type I intermediate filament protein primarily expressed in specialized epithelial cells. It has emerged as a significant biomarker in cancer research due to its differential expression patterns in various tumors. Studies have demonstrated that KRT17 is abnormally upregulated in multiple cancer types, including laryngeal squamous cell carcinoma (LSCC), where its elevated expression correlates with clinical stage, differentiation status, T classification, and lymph node metastasis . The protein appears to accelerate cell proliferation and invasion potential, suggesting its role as an oncogenic driver . KRT17's significance stems from its potential as both a diagnostic marker and therapeutic target, making KRT17 antibodies invaluable tools for cancer research and clinical applications.

What are the common host organisms for KRT17 antibodies and how does this affect research applications?

KRT17 antibodies are commonly produced in either mouse or rabbit host organisms, with each offering distinct advantages for specific applications:

The choice between mouse and rabbit hosts significantly impacts research applications. Mouse monoclonal antibodies (like clone E3) offer highly specific binding to particular epitopes, making them ideal for applications requiring precise target recognition . In contrast, rabbit polyclonal antibodies recognize multiple epitopes on the KRT17 protein, potentially providing stronger signals in applications like Western blotting and immunohistochemistry . Mouse monoclonals are particularly valuable when cross-reactivity is a concern, while rabbit polyclonals may offer advantages when signal amplification is necessary or when detecting KRT17 variants or modified forms.

What is the molecular weight of KRT17 and how can I verify antibody specificity?

The calculated molecular weight of KRT17 is 48 kDa, which corresponds to the observed molecular weight in experimental settings . When verifying antibody specificity, researchers should expect to detect a distinct band at approximately 48 kDa in Western blot analyses.

To confirm antibody specificity:

• Perform Western blot analysis using positive control samples known to express KRT17 (A431 cells, mouse skin tissue, or HeLa cells are recommended)

• Include negative controls (tissue or cell lines with low or no KRT17 expression)

• Validate results with knockdown/knockout models to demonstrate signal loss when KRT17 is depleted

• Compare staining patterns across multiple applications (WB, IHC, IF) to ensure consistent target recognition

• Consider using multiple antibodies targeting different KRT17 epitopes to confirm findings

The observed banding pattern should match the expected molecular weight, and signal intensity should correlate with known expression levels across different sample types. Antibody validation is crucial before proceeding with experimental studies to ensure reliable and reproducible results.

What are the optimal dilutions and conditions for KRT17 antibody in different applications?

The optimal working dilutions for KRT17 antibodies vary significantly depending on the specific application and the antibody formulation being used:

These recommendations serve as starting points, and optimal conditions should be determined empirically for each experimental system . For immunohistochemistry applications, antigen retrieval methods significantly impact staining quality, with TE buffer (pH 9.0) generally yielding better results for KRT17 detection, though citrate buffer (pH 6.0) can serve as an alternative . When working with formalin-fixed, paraffin-embedded tissues, extended antigen retrieval times may be necessary to expose KRT17 epitopes adequately. Additionally, blocking conditions and incubation times should be optimized to maximize signal-to-noise ratio across all applications.

How should KRT17 antibodies be stored and handled to maintain optimal activity?

Proper storage and handling of KRT17 antibodies are crucial for maintaining their activity and ensuring consistent experimental results:

• Storage temperature: Store at -20°C for long-term preservation. KRT17 antibodies formulated with glycerol (typically 50%) and PBS with 0.02% sodium azide are stable for one year after shipment when properly stored .

• Aliquoting: For antibodies stored at -20°C, aliquoting is generally unnecessary, though it may be beneficial for frequently used antibodies to prevent repeated freeze-thaw cycles .

• Working dilutions: Prepare working dilutions immediately before use and discard any unused diluted antibody. Working solutions should not be stored for extended periods.

• Freeze-thaw cycles: Minimize freeze-thaw cycles as they can lead to protein denaturation and loss of antibody activity. If the antibody must be repeatedly accessed, consider preparing small aliquots.

• Handling during experiments: Keep antibodies on ice or at 4°C during experiments to prevent degradation. Avoid exposure to strong light, particularly for fluorophore-conjugated antibodies.

• Contamination prevention: Use clean pipette tips and tubes to prevent cross-contamination and microbial growth which can degrade antibody performance.

Following these storage and handling guidelines will help ensure consistent antibody performance and reproducible experimental results across studies.

What controls should be included when using KRT17 antibodies in experimental studies?

Implementing appropriate controls is essential for ensuring the validity and reliability of experimental results with KRT17 antibodies:

Positive Controls:

• A431 cells, HeLa cells, and mouse skin tissue have been validated as positive controls for KRT17 expression

• Human cervical cancer tissue, lung cancer tissue, and Bowen's disease samples show robust KRT17 expression for IHC applications

• For tumor studies, samples with known high KRT17 expression based on previous research or database information

Negative Controls:

• Primary antibody omission control to assess non-specific binding of secondary antibodies

• Isotype control (using an irrelevant antibody of the same isotype) to evaluate non-specific binding

• Tissues or cell lines with confirmed low or absent KRT17 expression

• KRT17 knockout/knockdown samples, particularly important for validating antibody specificity

Procedural Controls:

• Loading controls for Western blot (β-actin, GAPDH, or total protein stains)

• Tissue controls with known staining patterns for IHC/IF

• Multiplexed staining with established markers to confirm cell/tissue identity

Validation Controls:

• Using multiple antibodies targeting different KRT17 epitopes to confirm findings

• Correlation between protein detection methods (e.g., comparing IHC results with Western blot findings)

• Pre-absorption with immunizing peptide to demonstrate specificity

Including these controls systematically will significantly enhance data quality and reproducibility when working with KRT17 antibodies across different experimental platforms.

How can KRT17 antibodies be used to study its role in cancer progression and metastasis?

KRT17 antibodies are powerful tools for investigating the protein's involvement in cancer progression through multiple experimental approaches:

• Expression correlation studies: KRT17 antibodies can be used in IHC analysis of tissue microarrays to correlate expression levels with clinicopathological parameters and patient outcomes. Research has demonstrated that KRT17 expression correlates significantly with tumor differentiation (P < 0.001), T classification (P < 0.01), lymph node metastasis (P < 0.05), and clinical stage (P < 0.05) in laryngeal squamous cell carcinoma .

• Functional studies:

  • Combine KRT17 antibodies with proliferation markers (Ki-67, PCNA) to assess correlation between KRT17 expression and cell proliferation

  • Use in cell migration/invasion assays following KRT17 knockdown/overexpression to evaluate its role in metastatic potential

  • Apply in co-immunoprecipitation experiments to identify interaction partners driving oncogenic signaling

• Mechanistic investigations:

  • Employ antibodies for chromatin immunoprecipitation (ChIP) assays to study KRT17's potential role in transcriptional regulation

  • Use in proximity ligation assays to visualize protein-protein interactions in situ

  • Apply in immunofluorescence co-localization studies to determine subcellular distribution changes during cancer progression

• Therapeutic development:

  • Screen for compounds that modulate KRT17 expression or function using antibody-based readouts

  • Develop antibody-drug conjugates targeting KRT17-expressing cancer cells

  • Monitor treatment response through quantitative assessment of KRT17 expression

These advanced applications of KRT17 antibodies can provide critical insights into the molecular mechanisms by which KRT17 contributes to cancer progression and potentially identify new therapeutic strategies for KRT17-overexpressing tumors.

How does KRT17 expression in different tumor types compare, and what techniques can best demonstrate these differences?

KRT17 expression varies significantly across tumor types, with several techniques available to accurately characterize these differences:

Comparative Expression Analysis:
Research utilizing multiple antibody-based detection methods has revealed differential KRT17 expression patterns across cancer types. The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) database analyses have established that KRT17 is significantly overexpressed in multiple cancer types compared to corresponding normal tissues . Particularly high expression has been observed in squamous cell carcinomas of various organs.

Optimal Detection Techniques:

• Immunohistochemistry (IHC):

  • Provides spatial context and cellular localization information

  • Enables semi-quantitative scoring of expression intensity (negative, weak, moderate, strong)

  • Allows assessment of cellular heterogeneity and tumor microenvironment

  • Optimal dilutions (1:4000-1:16000) with TE buffer pH 9.0 for antigen retrieval yield best results

• Multiplexed Immunofluorescence:

  • Enables co-expression analysis with other markers

  • Provides superior quantification capabilities

  • Allows for subcellular localization assessment

  • Recommended dilutions range from 1:300-1:1200

• Tissue Microarray Analysis:

  • Facilitates high-throughput comparison across multiple tumor types

  • Enables standardized staining conditions for reliable comparison

  • Allows correlation with clinical outcomes data

• Western Blotting:

  • Provides quantitative expression data

  • Confirms antibody specificity at the expected 48 kDa molecular weight

  • Effective at dilutions ranging from 1:1000-1:20000

When comparing KRT17 expression across tumor types, it's essential to use standardized scoring systems and consistent detection methodologies to enable reliable cross-study comparisons. The Human Protein Atlas approach, which classifies staining intensity into four categories (strong, moderate, weak, and negative) based on the proportion of stained cells, offers a robust framework for such comparisons .

What is the significance of KRT17 in immune cell infiltration and tumor microenvironment?

Recent research has uncovered important connections between KRT17 expression and immune cell infiltration in the tumor microenvironment, suggesting broader implications for cancer immunology:

• Immune Cell Correlation: Database analyses using tools like TIMER2.0 have revealed significant associations between KRT17 expression levels and the infiltration of specific immune cell populations in various tumor types . This correlation suggests KRT17 may influence the composition of the tumor immune microenvironment.

• Inflammatory Signaling: KRT17 has been implicated in modulating inflammatory pathways within the tumor microenvironment. It may participate in cytokine signaling networks that regulate immune cell recruitment and activation states.

• Prognostic Implications: The combined assessment of KRT17 expression and immune cell infiltration patterns may provide enhanced prognostic information beyond either marker alone. High KRT17 expression coupled with specific immune cell signatures could identify distinct patient subgroups with differential treatment responses.

• Immunotherapy Relevance: Understanding KRT17's relationship with immune cell infiltration could inform immunotherapy strategies. Patients with tumors expressing specific KRT17 patterns might respond differently to immune checkpoint inhibitors or other immunomodulatory therapies.

• Research Approaches:

  • Multiplex immunofluorescence using KRT17 antibodies alongside immune cell markers can visualize spatial relationships between KRT17-expressing tumor cells and infiltrating immune populations

  • FACS analysis using KRT17 antibodies in combination with immune cell markers can quantify associations

  • Single-cell approaches combining KRT17 detection with immune profiling can reveal heterogeneity within the tumor microenvironment

This emerging area of research suggests that KRT17 may have functions beyond its structural role in epithelial cells, potentially serving as an immunomodulatory factor in the tumor microenvironment with implications for therapeutic targeting and patient stratification.

What are common challenges when using KRT17 antibodies in FFPE tissues and how can they be overcome?

Working with KRT17 antibodies in formalin-fixed, paraffin-embedded (FFPE) tissues presents several challenges that can be addressed through methodological refinements:

Challenge 1: Insufficient antigen retrieval

• Solution: Optimize antigen retrieval conditions specifically for KRT17. Research indicates that TE buffer at pH 9.0 provides superior results compared to citrate buffer, though both can be effective . Extending retrieval time (20-30 minutes) at consistent temperature can improve epitope exposure in heavily fixed tissues.

Challenge 2: Variable fixation affecting epitope accessibility

• Solution: Standardize fixation protocols when possible. For historically collected samples with variable fixation, titrate antibody concentrations individually and consider dual antigen retrieval approaches (combining heat and enzymatic methods).

Challenge 3: High background staining

• Solution: Implement additional blocking steps using bovine serum albumin (BSA) or animal serum matching the secondary antibody host. Extend blocking time to 1-2 hours and include detergents like Triton X-100 (0.1-0.3%) to reduce non-specific binding.

Challenge 4: Weak or absent signal

• Solution: Employ signal amplification methods such as polymer-based detection systems or tyramide signal amplification. Reduce antibody dilution (staying within the 1:4000-1:16000 recommended range) and extend primary antibody incubation time (overnight at 4°C) .

Challenge 5: Inconsistent staining across tissue regions

• Solution: Ensure complete deparaffinization and rehydration. Consider using automated staining platforms to maintain consistent reagent delivery, temperature, and timing across the entire tissue section.

Challenge 6: Distinguishing true KRT17 signal from autofluorescence in IF applications

• Solution: Include autofluorescence quenching steps (such as Sudan Black B treatment) before antibody application. Utilize spectral imaging and unmixing techniques to separate true signal from tissue autofluorescence.

Implementation of these troubleshooting approaches can significantly improve the quality and reliability of KRT17 detection in FFPE tissues across both research and clinical diagnostic applications.

How can researchers validate knockdown or knockout of KRT17 in experimental models?

Validating successful KRT17 knockdown or knockout is critical for functional studies and can be accomplished through multiple complementary approaches:

1. Protein-level validation:

• Western Blot Analysis: Using validated KRT17 antibodies at dilutions of 1:1000-1:20000 to detect the 48 kDa KRT17 protein . Quantification should show significant reduction in KRT17 signal compared to controls.

• Immunocytochemistry/Immunofluorescence: Applied at 1:300-1:1200 dilutions to visualize reduction or absence of KRT17 in individual cells, providing information about knockdown efficiency at the single-cell level .

• Flow Cytometry: Particularly useful for quantifying knockdown efficiency across cell populations and identifying potential subpopulations with differential knockdown success.

2. mRNA-level validation:

• RT-qPCR: To quantify reduction in KRT17 transcript levels, providing complementary evidence to protein-level changes.

• RNA-Seq: For comprehensive transcriptomic analysis to confirm KRT17 reduction and assess potential compensatory changes in related genes.

3. Genomic validation (for knockout models):

• PCR genotyping: To confirm genetic modifications at the KRT17 locus.

• Sequencing: To verify the exact nature of the introduced genetic modification.

4. Functional validation:

• Phenotypic assays: Assess changes in cellular properties known to be influenced by KRT17, such as proliferation rates or invasive capacity in cancer cell models .

• Rescue experiments: Re-introduction of KRT17 to knockout/knockdown models should restore the original phenotype if changes are specifically due to KRT17 loss.

5. Controls to include:

• Non-targeting siRNA/shRNA controls for knockdown studies

• Wild-type parental cells for knockout models

• Multiple independent knockdown/knockout clones to control for off-target effects

• Positive control samples with known KRT17 expression (A431 cells, HeLa cells)

The combination of these validation approaches provides robust confirmation of successful KRT17 manipulation and strengthens the reliability of subsequent functional studies investigating KRT17's biological roles.

What are the best approaches for quantifying KRT17 expression in tissue samples for clinical correlations?

Accurate quantification of KRT17 expression in tissue samples is essential for establishing meaningful clinical correlations. Several complementary approaches can be implemented:

1. Immunohistochemistry with Digital Image Analysis:

• Apply KRT17 antibodies at optimized dilutions (1:4000-1:16000) with appropriate antigen retrieval

• Capture high-resolution whole slide images

• Implement digital image analysis software to quantify:

  • Percentage of positive cells

  • Staining intensity (0-3+ scale)

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

  • Tumor heterogeneity assessment

• Advantages: Preserves tissue architecture context, applicable to routine clinical samples

2. Multiplexed Protein Quantification:

• Multiplex immunofluorescence with KRT17 and relevant biomarkers

• Use automated multispectral imaging systems

• Quantify co-expression patterns and spatial relationships

• Advantages: Provides contextual information about KRT17 in relation to other markers

3. Tissue Microarray (TMA) Analysis:

• Construct TMAs with tumor and matched normal tissues

• Apply standardized IHC protocols across multiple samples

• Score using validated systems (e.g., Human Protein Atlas approach classifying staining as strong, moderate, weak, or negative)

• Advantages: High throughput, reduced technical variation

4. Molecular Methods for Absolute Quantification:

• Targeted mass spectrometry for precise protein quantification

• RT-qPCR for mRNA level assessment

• Digital droplet PCR for absolute transcript quantification

• Advantages: Higher precision and reproducibility

5. Pathologist-based Scoring Systems:

• Semi-quantitative assessment by trained pathologists

• Implementation of weighted scoring incorporating:

  • Staining intensity

  • Percentage of positive cells

  • Subcellular localization patterns

• Advantages: Integrates expert interpretation of complex staining patterns

6. Statistical Approaches for Clinical Correlation:

• Categorical analysis (high vs. low expression using established cutoffs)

• Continuous variable analysis (correlation between expression levels and clinical parameters)

• Multivariate analysis adjusting for confounding clinicopathological variables

• Survival analysis using Kaplan-Meier and Cox regression methods

Research has demonstrated that KRT17 expression correlates significantly with differentiation (P < 0.001), T classification (P < 0.01), lymph node metastasis (P < 0.05), and clinical stage (P < 0.05) in laryngeal squamous cell carcinoma , highlighting the importance of robust quantification approaches for establishing clinically relevant associations.

How is KRT17 being explored as a therapeutic target in cancer, and what antibody-based approaches are being developed?

KRT17's emerging role as a potential therapeutic target in cancer has sparked several innovative antibody-based approaches:

• Antibody-Drug Conjugates (ADCs):

  • Development of KRT17-targeted ADCs leveraging the elevated expression in multiple tumor types

  • Selection of highly specific monoclonal antibodies coupled with potent cytotoxic payloads

  • Challenges include optimizing antibody internalization rates and managing potential off-target effects

• CAR-T Cell Therapy:

  • Engineering of chimeric antigen receptor T cells targeting KRT17-expressing tumor cells

  • Requires highly specific single-chain variable fragments derived from validated KRT17 antibodies

  • Addressing challenges of targeting an intracellular protein through MHC-presented epitopes

• Blocking Antibodies for Protein-Protein Interactions:

  • Development of antibodies that disrupt KRT17 interactions with signaling partners

  • Focus on epitopes involved in key protein-protein interactions that drive oncogenic signaling

  • Requires detailed understanding of KRT17's structural domains and binding interfaces

• Bi-specific Antibodies:

  • Creation of bi-specific constructs linking KRT17 recognition with immune cell recruitment

  • Potential to enhance immune surveillance of KRT17-overexpressing tumors

  • Addresses challenges of tumor microenvironment immunosuppression

• Diagnostic-Therapeutic Combinations:

  • Integration of KRT17 antibody diagnostics with targeted therapeutics

  • Development of companion diagnostics to identify patients most likely to benefit from KRT17-targeted therapies

  • Implementation of immunoPET approaches using radiolabeled KRT17 antibodies for theranostic applications

These approaches are still in early developmental stages, with research ongoing to address critical questions about specificity, efficacy, and potential resistance mechanisms. The reported correlation between KRT17 expression and clinical parameters in cancers like LSCC provides strong rationale for continuing to explore its potential as a therapeutic target.

What are the latest findings regarding post-translational modifications of KRT17 and how can researchers study them?

Recent research has uncovered important roles for post-translational modifications (PTMs) of KRT17 in regulating its function and involvement in disease processes:

Key Post-translational Modifications of KRT17:

• Phosphorylation:

  • Occurs primarily on serine/threonine residues

  • Regulates KRT17 solubility, filament assembly, and protein-protein interactions

  • Often modulated during stress responses and cell cycle progression

• Glycosylation:

  • O-GlcNAcylation may influence KRT17 stability and subcellular localization

  • Potentially altered in cancer cells with dysregulated glucose metabolism

• Ubiquitination:

  • Regulates KRT17 turnover and degradation

  • May be dysregulated in pathological conditions with abnormal KRT17 accumulation

• Acetylation/Deacetylation:

  • Impacts KRT17's binding properties and transcriptional regulatory functions

  • Often responsive to cellular metabolic state

Methodologies for Studying KRT17 PTMs:

• Modification-Specific Antibodies:

  • Development of antibodies recognizing specific PTM sites on KRT17

  • Application in Western blotting, IHC, and IF to study PTM patterns in tissues and cells

  • Validation through PTM-blocking peptides and site-directed mutagenesis

• Mass Spectrometry Approaches:

  • Phosphoproteomic analysis to identify KRT17 phosphorylation sites

  • Glycoproteomics to characterize glycosylation patterns

  • Quantitative MS to determine stoichiometry of modifications

• Site-Directed Mutagenesis:

  • Generation of KRT17 variants with modified PTM sites (phosphomimetic or phosphodeficient)

  • Functional characterization to determine impact on KRT17 properties

  • Expression in KRT17-knockout backgrounds to assess phenotypic rescue

• Proximity Ligation Assays:

  • Detection of specific KRT17 PTMs in situ

  • Visualization of co-occurrence with interacting proteins

  • Assessment of spatial distribution within cells and tissues

• Pharmacological Modulation:

  • Application of kinase/phosphatase inhibitors to manipulate KRT17 phosphorylation

  • Use of deacetylase inhibitors to study acetylation effects

  • Assessment of proteasome inhibitors on ubiquitinated KRT17 forms

Understanding KRT17 PTMs is particularly relevant given its emerging roles beyond structural functions, including potential involvement in signaling pathways relevant to cancer progression and immune regulation . Research in this area may reveal new therapeutic opportunities targeting specific modified forms of KRT17.

How do different KRT17 antibody clones compare in their ability to detect specific KRT17 isoforms or modified forms?

The selectivity of different KRT17 antibody clones for specific isoforms or modified forms represents a critical consideration for advanced research applications:

Comparison of Major KRT17 Antibody Clones:

Detection of Modified Forms:

• Phosphorylated KRT17:

  • Polyclonal antibodies generally show superior detection of phosphorylated forms due to epitope diversity

  • Phospho-specific antibodies are necessary for targeted research on specific modification sites

  • Epitope masking due to phosphorylation can reduce binding efficiency of some monoclonal antibodies

• Proteolytically Processed Forms:

  • C-terminal-directed antibodies like SPM560 may fail to detect truncated forms

  • N-terminal antibodies could miss C-terminal fragments with biological activity

  • Combining antibodies targeting different regions provides comprehensive detection

• Cross-reactivity Considerations:

  • Sequence homology between KRT17 and other type I keratins requires careful validation

  • Monoclonals like E3 offer higher specificity for distinguishing KRT17 from similar keratins

  • Validation in knockout/knockdown systems is essential for confirming isoform specificity

• Application-specific Performance:

  • Some clones perform better in native conditions (IF/IHC) than denaturing conditions (WB)

  • Fixation methods can differentially affect epitope accessibility for various clones

  • Antibody selection should be guided by the specific experimental question and technique