KRT19 Antibody

What is KRT19 and why is it important in cancer research?

KRT19 (also known as Cytokeratin 19 or CK19) is the smallest known type I intermediate filament protein, consisting of 400 amino acids with a molecular weight of approximately 44.1 kDa. It functions as a structural constituent of muscle and cytoskeleton and plays important roles in development. KRT19 serves as a valuable cancer marker, particularly for the detection of disseminated tumors in lymph nodes, peripheral blood, and bone marrow. This protein is expressed in various epithelial tissues including sweat gland, mammary gland ductal cells, bile ducts, gastrointestinal tract, bladder urothelium, oral epithelia, esophagus, and ectocervical epithelium . Recent research has revealed that KRT19 plays crucial regulatory roles in cancer progression through various signaling pathways, making it an important target for both diagnostic and therapeutic applications .

What are the technical specifications of KRT19 and available antibodies?

KRT19 is a 44.1 kDa intermediate filament protein that serves as a structural component in epithelial cells. Various KRT19 antibodies are available for research applications:

Recommended starting concentrations include 2-5 μg/ml for mouse monoclonal antibodies in IHC and ICC applications, and 0.2-0.5 μg/ml for rabbit antibodies, which generally demonstrate greater affinity . For Western blots, concentrations of 0.2-0.5 μg/ml for mouse antibodies and 20-50 ng/ml for rabbit antibodies are suggested as initial testing points .

How do KRT19 expression patterns differ across cancer types?

KRT19 expression varies significantly across different cancer types, with important implications for both diagnosis and prognosis:

• Breast cancer: KRT19 is frequently upregulated and plays a role in enhancing cancer properties through interaction with HER2 signaling and Notch pathway activation .

• Colon cancer: While KRT19 is overexpressed, it demonstrates an opposite functional effect compared to breast cancer. In colon cancer, KRT19 promotes tumorigenesis via upregulation of Wnt signaling and downregulation of Notch signaling .

• Thyroid cancer: KRT19 serves as a particularly useful marker for papillary thyroid carcinoma, with approximately 50-60% of follicular carcinomas also showing positive labeling .

Interestingly, survival analysis has shown that high KRT19 expression is associated with better outcomes in breast cancer but poorer outcomes in lung and ovarian cancers . These contradictory roles of KRT19 in different cancer types highlight the importance of tissue-specific context in understanding KRT19 function and utilizing KRT19 antibodies for diagnostic purposes.

What are the optimal sample preparation techniques for KRT19 antibody applications?

Different applications require specific sample preparation techniques to achieve optimal KRT19 detection:

For immunohistochemistry (IHC):

• Formalin-fixed paraffin-embedded (FFPE) tissues require antigen retrieval

• For paraffin sections, a Tris-EDTA buffer at pH 9.0 has shown successful results

• Recommended antibody dilutions range from 1:20 to 1:2500, depending on the specific antibody and detection system

For immunocytochemistry (ICC):

• Methanol:acetone (1:1) fixation works well for cultured cells

• For immunofluorescence applications, optimal antibody concentrations generally range from 0.25-2 μg/ml

For flow cytometry:

• Cell fixation and permeabilization using specialized buffers like FlowX FoxP3 Fixation & Permeabilization Buffer improves intracellular staining

• Both direct and indirect labeling approaches can be used, with conjugated antibodies offering advantages for multicolor analysis

For Western blotting:

• Standard protein extraction methods are generally effective for KRT19 detection

• The protein runs at approximately 40-44 kDa on SDS-PAGE gels

These optimized protocols ensure reliable KRT19 detection across various experimental platforms while minimizing background and non-specific signals.

How should KRT19 antibodies be validated for research applications?

Rigorous validation of KRT19 antibodies is essential for generating reliable research data. A comprehensive validation approach should include:

• Expression verification: Confirm antibody reactivity in tissues/cells with known KRT19 expression (positive controls) such as epithelial cells from breast, colon, or thyroid tissues.

• Knockdown experiments: Use siRNA or shRNA against KRT19 to demonstrate reduced or abolished signal with the antibody. Multiple studies have validated KRT19 antibodies using this approach, showing reduced immunoreactivity after KRT19 knockdown .

• Multiple detection methods: Cross-validate antibody performance across different applications (Western blot, IHC, ICC) to ensure consistent results.

• Orthogonal validation: Compare antibody results with mRNA expression data (RNAseq or qPCR) to confirm correlation between protein and transcript levels .

• Epitope mapping: Understanding the specific region of KRT19 recognized by the antibody can help interpret results, especially when studying post-translational modifications or protein interactions .

• Cross-reactivity assessment: Test antibody specificity against other keratins, particularly closely related type I keratins, to confirm target specificity.

Enhanced validation approaches, such as the orthogonal RNAseq validation mentioned for some commercial antibodies, provide additional confidence in antibody specificity and performance .

What criteria should guide KRT19 antibody selection for specific research applications?

Selection of the appropriate KRT19 antibody should be guided by several key considerations:

For diagnostic applications:

• Monoclonal antibodies offer high specificity and reproducibility, making them ideal for standardized diagnostic assays

• Antibodies targeting conserved epitopes ensure consistent detection across patient samples

• Validated antibodies with established performance in clinical settings should be prioritized

For mechanistic studies:

• Consider antibodies targeting specific domains or post-translational modifications

• For studies examining KRT19 phosphorylation by Akt, antibodies that don't interfere with the Ser35 region may be preferred

• For protein interaction studies, epitope location should not overlap with binding regions

For multi-color imaging:

• Pre-conjugated antibodies eliminate cross-reactivity from secondary antibodies

• Note that blue fluorescent dye conjugates (CF®405S, CF®405M) are not recommended for detecting low abundance targets due to lower fluorescence and potentially higher background

For quantitative applications:

• Linearity of signal should be validated across a range of protein concentrations

• Consistent lot-to-lot performance is essential for longitudinal studies

The specific experimental question, detection method, and sample type should all factor into antibody selection to ensure optimal results and data reliability.

How does KRT19 interact with HER2 signaling in breast cancer?

KRT19 demonstrates a complex and significant relationship with HER2 signaling in breast cancer through several mechanisms:

• Expression regulation: HER2 signaling upregulates KRT19 expression in breast cancer cells through downstream ERK activity at the transcriptional level .

• Post-translational modification: Another HER2-downstream kinase, Akt, phosphorylates KRT19 specifically at Ser35. This phosphorylation triggers significant changes in KRT19 structure and localization, converting it from a filamentous to a granulous form and inducing membrane translocation .

• Protein stabilization: Phosphorylated KRT19 plays a critical role in stabilizing HER2 protein. It binds directly to HER2 on the plasma membrane and inhibits proteasome-mediated degradation of HER2. This creates a positive feedback loop where HER2 signaling enhances KRT19 expression and modification, which in turn stabilizes HER2 .

• Ubiquitination regulation: Silencing of KRT19 by shRNA results in increased ubiquitination and destabilization of HER2. This effect is dependent on the phosphorylation status of KRT19, as mutation of the Ser35 residue eliminates the ability of KRT19 to prevent HER2 ubiquitination .

This intricate relationship between KRT19 and HER2 has significant therapeutic implications, as targeting KRT19 with antibodies has been shown to downregulate HER2 and reduce cancer cell viability, even in Trastuzumab-resistant cell lines .

Why does KRT19 demonstrate opposing effects in different cancer types?

One of the most intriguing aspects of KRT19 biology is its contradictory roles in different cancer types. Research has revealed that:

In breast cancer:

• KRT19 knockdown enhances cancer properties

• Silencing KRT19 leads to attenuated Wnt signaling and enhanced Notch signaling

• KRT19 interacts with the β-catenin/RAC1 complex

• This interaction leads to upregulation of NUMB, a negative regulator of Notch signaling

In colon cancer:

• KRT19 knockdown suppresses cancer properties

• Silencing KRT19 results in downregulation of Wnt/Notch signaling

• KRT19 interacts with β-catenin but not with RAC1

• This allows LEF/TCF transcription factors to bind primarily to LEF1 and TCF7 promoter regions

These tissue-specific molecular interactions explain why KRT19 can function as both a tumor promoter or suppressor depending on cellular context. Survival analysis confirms these opposing effects, showing that high KRT19 expression is associated with better outcomes in breast cancer (hazard ratio = 0.83) but poorer outcomes in lung cancer (hazard ratio = 1.31) .

This complexity underscores the importance of understanding tissue-specific KRT19 functions when developing diagnostic or therapeutic approaches targeting this protein.

How can KRT19 expression be integrated with immune markers for improved cancer prognosis?

Recent research has revealed significant correlations between KRT19 expression and immune cell infiltration in breast cancer, offering new approaches to cancer prognosis:

These findings suggest that combining KRT19 expression analysis with immune infiltration assessment could become a powerful new prognostic approach for breast cancer, potentially guiding immunotherapy decisions and improving patient stratification.

How do post-translational modifications of KRT19 affect antibody binding and experimental design?

Post-translational modifications (PTMs) of KRT19, particularly phosphorylation, present important considerations for antibody selection and experimental design:

• Phosphorylation at Ser35: KRT19 is phosphorylated by Akt specifically at Ser35, as confirmed through mutation studies and in vitro kinase assays . This phosphorylation dramatically alters KRT19's structure and function, inducing membrane translocation and enabling HER2 binding.

• Antibody epitope considerations: When studying phosphorylated KRT19, researchers must consider whether the antibody epitope includes or is adjacent to the Ser35 region. Some antibodies may show differential binding to phosphorylated versus non-phosphorylated forms.

• Phospho-specific detection: For studies focused on the phosphorylated form of KRT19, specialized approaches may be needed:

  • Phospho-specific antibodies (such as those recognizing phosphorylated RXX(S/T) residues)

  • Akt substrate antibodies can detect phosphorylated KRT19 after immunoprecipitation

  • Treatment with phosphatases before antibody detection can confirm phosphorylation-dependent signals

• Subcellular localization studies: Standard KRT19 antibodies may detect different pools of the protein depending on its phosphorylation state. When examining membrane-localized KRT19, specialized techniques are recommended:

  • Cell surface biotinylation assays

  • Cell fractionation followed by Western blotting

  • Cell surface immunofluorescence labeling

Understanding these technical considerations is essential for accurately interpreting KRT19 antibody results, particularly in mechanistic studies examining the protein's role in cancer signaling pathways.

What therapeutic potential do KRT19 antibodies demonstrate in preclinical cancer models?

KRT19 antibodies have shown promising therapeutic effects in preclinical cancer models, particularly for HER2-positive cancers:

• Anti-proliferative effects: Treatment with KRT19 antibodies demonstrated a dose-dependent and time-dependent decrease in proliferation of HER2-positive cancer cells in vitro .

• Mechanism of action: The therapeutic effect occurs through:

  • Increased ubiquitination of HER2

  • Enhanced proteasome-mediated degradation of HER2

  • Downregulation of HER2 protein levels and subsequent reduction in downstream signaling

• Effectiveness in resistant models: Notably, KRT19 antibody treatment was effective against Trastuzumab-resistant JIMT-1 cells, suggesting potential for addressing therapy resistance .

• In vivo efficacy: In xenograft experiments, treatment with KRT19 antibody inhibited tumor formation:

  • Complete tumor dissipation was observed in all mice treated with KRT19 antibody

  • The effect was comparable to Trastuzumab treatment

  • Administration appeared non-toxic based on animal survival and weight analysis

These findings suggest KRT19 antibodies could represent a novel therapeutic approach for HER2-positive cancers, potentially including those resistant to current HER2-targeted therapies. The unique mechanism—disrupting the KRT19-HER2 interaction rather than directly targeting HER2—provides a new strategy for addressing HER2-dependent cancers.

How can KRT19 antibodies be used to distinguish cancer subtypes with differential prognosis?

KRT19 antibodies serve as valuable tools for cancer subtyping and prognostic stratification:

• Thyroid cancer classification:

  • KRT19 antibodies are particularly useful in identifying papillary thyroid carcinoma

  • Approximately 50-60% of follicular carcinomas also show positive labeling

  • The staining pattern and intensity can help distinguish between aggressive and less aggressive variants

• Breast cancer subtyping:

  • KRT19 expression patterns differ across breast cancer molecular subtypes

  • The relationship between KRT19 and HER2 makes KRT19 staining particularly relevant for HER2-positive breast cancers

  • Combined analysis of KRT19 with HER2 and hormone receptors improves prognostic accuracy

• Gastrointestinal tumor differentiation:

  • KRT19 antibodies help characterize adenocarcinomas of the colon, stomach, pancreas, and biliary tract

  • The staining pattern can assist in distinguishing primary vs. metastatic tumors

• Integration with immune markers:

  • As shown in recent research, combining KRT19 expression analysis with immune cell markers (CD8+ T cells, B cells, macrophages) provides enhanced prognostic information

  • This integrated approach enables more accurate patient stratification and potentially guides treatment decisions

The prognostic value of KRT19 varies by cancer type—associated with better outcomes in breast cancer (hazard ratio = 0.83) but worse outcomes in lung cancer (hazard ratio = 1.31) and potentially ovarian cancer (hazard ratio = 1.09) . These tissue-specific differences highlight the importance of contextual interpretation of KRT19 immunostaining in clinical applications.

What are common technical challenges in KRT19 immunostaining and how can they be addressed?

Researchers frequently encounter several technical challenges when performing KRT19 immunostaining:

• Background staining issues:

  • Problem: Non-specific cytoplasmic staining in negative tissues

  • Solution: Optimize blocking conditions (5-10% normal serum from secondary antibody species); use more dilute antibody concentrations; include additional washing steps with 0.1% Tween-20 in PBS

• Antigen retrieval effectiveness:

  • Problem: Weak or inconsistent staining in FFPE tissues

  • Solution: For paraffin sections, use Tris-EDTA buffer at pH 9.0, which has proven successful for KRT19 antibodies ; perform heat-induced epitope retrieval at optimal temperature and duration (typically 95-100°C for 20-30 minutes)

• Cross-reactivity with other keratins:

  • Problem: Detection of similarly sized keratins, causing specificity concerns

  • Solution: Select monoclonal antibodies with validated specificity; use antibodies targeting unique regions of KRT19; include appropriate controls including KRT19 knockdown samples

• Membrane vs. cytoplasmic detection:

  • Problem: Difficulty detecting membrane-localized KRT19, especially in HER2-positive cancers

  • Solution: Use specialized protocols like cell surface immunofluorescence labeling or cell-surface protein biotinylation assays as demonstrated in published studies ; consider membrane protein extraction methods

• Fixation method variability:

  • Problem: Different fixatives affect KRT19 antibody performance

  • Solution: For cultured cells, methanol:acetone (1:1) fixation has shown good results ; standardize fixation protocols within studies; include properly fixed positive control samples

Proper experimental design with appropriate controls (both positive and negative) is essential for troubleshooting these issues and ensuring reliable, reproducible KRT19 detection.

How can researchers resolve discrepancies between KRT19 antibody results and other detection methods?

When facing discrepancies between KRT19 antibody results and other detection methods, researchers should consider:

• Transcript vs. protein level discrepancies:

  • KRT19 protein may be regulated post-transcriptionally, leading to differences between mRNA and protein detection

  • Validate with multiple antibodies targeting different epitopes

  • Consider protein stability and half-life differences across experimental conditions

• Antibody epitope accessibility issues:

  • Post-translational modifications like phosphorylation at Ser35 can alter KRT19 structure and epitope exposure

  • Protein-protein interactions, particularly with HER2 or β-catenin, may mask antibody binding sites

  • Try denaturing conditions for Western blots or alternative fixation methods for IHC/ICC

• Isoform-specific detection:

  • Ensure the detection method (primers or antibodies) recognizes all relevant KRT19 isoforms

  • Cross-reference transcript variant information with antibody epitope mapping

• Technical considerations for resolution:

  • When mRNA and protein levels differ, assess protein degradation or stability effects

  • For contradictory antibody results, compare monoclonal vs. polyclonal antibodies

  • In cell lines showing inconsistent results, verify cell line authentication and passage number

  • For clinical samples, stratify by cancer subtype, as KRT19 has opposing effects in different cancer types

• Reconciliation strategies:

  • Orthogonal validation using multiple detection methods

  • Consider context-specific biology, as KRT19 functions differently across tissues

  • Evaluate subcellular localization carefully, as phosphorylated KRT19 redistributes from cytoplasmic to membrane locations

Understanding the biological complexity of KRT19 regulation and function can help explain apparent discrepancies and lead to more accurate interpretation of experimental results.