KRT19 Monoclonal Antibody

Basic Properties and Applications

• What is KRT19 and why is it significant in cancer research?

  KRT19 (Cytokeratin 19) is the smallest (40 kDa) member of the acidic type I cytokeratin family proteins. It possesses a highly preserved α-helical central domain but lacks the C-terminal non-helical tail domain typically found in other keratins. As a cytoplasmic intermediate filament protein, KRT19 provides structural rigidity, acts as a multipurpose scaffold, and serves as a marker of epithelial cells and tissues. In cancer research, KRT19 has gained significance as a biomarker for detecting disseminated tumor cells in bone marrow, lymph nodes, and peripheral blood of cancer patients, particularly in breast cancer. Its expression patterns correlate with clinical outcomes and cancer progression in multiple tumor types, making it valuable for both diagnostic and prognostic applications .

• What applications are KRT19 monoclonal antibodies validated for in research?

  KRT19 monoclonal antibodies have been validated for multiple research applications including:

  • Immunohistochemistry on paraffin-embedded tissues (IHC-P)

  • Immunocytochemistry (ICC)

  • Western blotting (WB)

  • Flow cytometry

  • Reverse transcriptase PCR-mediated detection of disseminated tumors

  These antibodies have been specifically validated with human and rat samples, with applications demonstrated in tissues such as breast adenocarcinoma cell lines (MCF7), colon tissue, and lung lysates . The antibodies are particularly useful in tumor detection and characterization of epithelial tissues in both normal and pathological conditions.

• What are the most common tissue types where KRT19 expression has been documented?

  KRT19 expression has been documented in several tissues and cell types including:

  • Epithelial tissues (particularly simple and stratified epithelia)

  • Breast tissue (with differential expression between normal and cancerous tissue)

  • Ovarian tissue (significantly upregulated in ovarian cancer compared to normal controls)

  • Hepatocellular carcinoma

  • Colon tissue

  • Muscle tissue (where it plays a role in organizing myofibers)

  Tissue-specific expression patterns make KRT19 particularly valuable as a differential marker for distinguishing cancerous from normal tissue in various organ systems . In cancer tissues, expression levels often correlate with aggressiveness and clinical outcomes.

Experimental Design and Methodology

• What are the optimal fixation and sample preparation methods when using KRT19 antibodies for immunohistochemistry?

  For optimal results with KRT19 antibodies in immunohistochemistry:

  • Formalin fixation and paraffin embedding (FFPE) have been validated for tissue samples

  • For cell lines, methanol (MeOH) fixation has shown good results, as demonstrated with MCF7 breast adenocarcinoma cells

  • Standard antigen retrieval techniques (heat-induced epitope retrieval in citrate buffer) are generally effective

  • For immunocytochemistry, a dilution of approximately 1 μg/ml has been effective for rat colon tissue

  • Secondary antibody selection should be compatible with the host species of the primary antibody (e.g., goat anti-mouse IgG-CF488 for mouse monoclonal KRT19 antibodies)

  • Nuclear counterstaining can be performed with standard DNA stains

  These protocols have been validated for specific antibodies like ab220193, with clear membrane and cytoplasmic staining patterns observed in positive samples .

• How should researchers design experiments to study KRT19's role in cancer cell migration and invasion?

  To study KRT19's role in cancer cell migration and invasion, researchers should consider the following experimental approach:

  • Gene Modulation: Utilize short hairpin RNA (shRNA) systems to silence KRT19 expression or overexpression vectors to increase KRT19 levels. Validate knockdown or overexpression by Western blot analysis and immunocytochemistry.

  • Functional Assays:

    • Cell proliferation assays (e.g., MTT, BrdU incorporation)

    • Migration assays (wound healing/scratch assays, transwell migration)

    • Invasion assays (Matrigel-coated transwell chambers)

    • Colony formation assays to assess anchorage-dependent growth

    • Cell-cycle analysis using flow cytometry

  • Signaling Pathway Analysis: Examine effects on the Akt signaling pathway, PTEN expression, and Egr1 nuclear localization through Western blotting and immunofluorescence.

  • In Vivo Validation: Consider mouse xenograft models to confirm in vitro findings about KRT19's role in tumor formation and growth.

  Previous studies have demonstrated that silencing KRT19 resulted in increased cell proliferation, migration, invasion, and survival through upregulation of Akt signaling and reduced PTEN mRNA expression .

• What are the recommended controls when using KRT19 antibodies in cancer research?

  When using KRT19 antibodies in cancer research, researchers should include the following controls:

  • Positive Controls:

    • MCF7 cells (human breast adenocarcinoma cell line) expressing high levels of KRT19

    • Known KRT19-positive tissues (specific to species being studied)

    • For breast cancer research, luminal A breast cancer tissues typically express KRT19

  • Negative Controls:

    • Isotype-matched irrelevant antibody controls

    • Cell lines with confirmed low/no KRT19 expression

    • Triple-negative breast cancer cells like MDA-MB231 may show lower KRT19 expression than luminal types

    • Secondary antibody-only controls to assess non-specific binding

  • Knockdown/Knockout Controls:

    • Cells with KRT19 knockdown via shRNA for antibody validation

    • Tissues from KRT19 knockout animals when available

  • Expression Gradient Controls:

    • Include samples with varying expression levels to demonstrate antibody sensitivity and dynamic range

  These controls help validate antibody specificity and ensure reliable interpretation of experimental results .

Technical Troubleshooting

• What are common technical challenges when working with KRT19 antibodies and how can they be addressed?

  Researchers commonly encounter several technical challenges when working with KRT19 antibodies:

  • Background Staining Issues:

    • Problem: Non-specific background staining in IHC or ICC

    • Solution: Optimize blocking conditions (use 5-10% serum from the species of the secondary antibody); include 0.1-0.3% Triton X-100 for better antibody penetration; test different antibody dilutions (start with 1 μg/ml for IHC-P)

  • Variable Expression Levels:

    • Problem: Inconsistent staining intensity across samples

    • Solution: Standardize fixation times; use positive and negative controls with each batch; consider antigen retrieval optimization; quantify results using image analysis software

  • Cross-Reactivity Concerns:

    • Problem: Potential cross-reactivity with other keratins

    • Solution: Validate specificity with Western blotting; use monoclonal antibodies with documented specificity (e.g., KRT19/800 clone); include appropriate knockdown controls

  • Sample Preparation Issues:

    • Problem: Loss of antigenicity during fixation

    • Solution: For FFPE tissues, limit fixation time to 12-24 hours; for ICC, methanol fixation has shown good results with MCF7 cells as demonstrated in the literature

  • Detection Sensitivity:

    • Problem: Weak signal in samples with low expression

    • Solution: Consider amplification systems (e.g., tyramide signal amplification); optimize primary antibody incubation time and temperature (overnight at 4°C may improve sensitivity)

  Each of these approaches has been validated in research settings and can significantly improve the reliability and reproducibility of experiments using KRT19 antibodies .

• How can researchers distinguish between KRT19 and other cytokeratin family members in experimental settings?

  Distinguishing between KRT19 and other cytokeratin family members requires careful consideration of several experimental approaches:

  • Antibody Selection:

    • Use monoclonal antibodies targeted to unique epitopes of KRT19 (KRT19/800 clone has demonstrated specificity)

    • Verify antibody specificity using Western blot analysis showing the expected molecular weight (40 kDa for KRT19)

    • Cross-reference antibody validation data for cross-reactivity with other keratins

  • Molecular Techniques:

    • Design PCR primers targeting unique regions of KRT19 mRNA

    • Employ RT-qPCR with primers designed to span exon-exon junctions specific to KRT19

    • Consider RNA-seq for comprehensive profiling of keratin expression

  • Protein Analysis:

    • Use 2D gel electrophoresis to separate keratins based on both molecular weight and isoelectric point

    • Perform mass spectrometry to identify specific keratin peptide sequences

    • Employ co-immunoprecipitation with KRT19-specific antibodies followed by mass spectrometry

  • Functional Validation:

    • Utilize KRT19 knockdown/knockout systems to confirm specificity of signals

    • Compare expression patterns across tissues known to differentially express KRT19 versus other keratins

    • Correlate findings with known functional attributes of KRT19 (lack of C-terminal tail domain, etc.)

  These approaches help ensure accurate identification of KRT19 specifically, reducing the likelihood of misattribution of experimental results to the wrong keratin family member .

Emerging Research Directions

• What is the current evidence for using KRT19 as a therapeutic target in cancer treatment?

  Emerging evidence suggests several promising avenues for targeting KRT19 therapeutically:

  • Cancer Stem Cell Modulation:

    • KRT19 regulates cancer stem cell properties through modulation of stem cell markers (ALDH1, CXCR4, CD133)

    • Potential for developing therapies that increase KRT19 expression in aggressive cancer types to attenuate stem cell-like properties and enhance drug sensitivity

    • In breast cancer models, overexpression of KRT19 in cancer stem-like cells resulted in significant attenuation of cancer properties

  • Immune Modulation Strategies:

    • Strong correlation between KRT19 expression and immune cell infiltration suggests immunotherapy applications

    • Combined KRT19 status assessment with immune profiling could identify patients most likely to benefit from immunotherapy

    • Patients with specific KRT19/immune infiltration patterns may be candidates for targeted immune approaches

  • Signaling Pathway Intervention:

    • KRT19's regulation of the Akt pathway through PTEN/Egr1 suggests potential for combination therapies targeting these pathways

    • Drugs affecting the nuclear import of Egr1 might reverse the effects of low KRT19 expression

    • Targeted approaches to restore PTEN expression could counteract the effects of KRT19 downregulation

  • Diagnostic/Therapeutic Combinations:

    • Development of KRT19-targeted delivery systems for cancer drugs

    • Potential for theranostic approaches using KRT19 for both detection and treatment

  While promising, these therapeutic approaches remain largely in preclinical development stages, requiring further validation before clinical translation .

• How might single-cell analysis technologies advance our understanding of KRT19's role in tumor heterogeneity?

  Single-cell analysis technologies offer powerful new approaches to understand KRT19's role in tumor heterogeneity:

  • Cellular Subpopulation Identification:

    • Single-cell RNA sequencing can identify distinct subpopulations within tumors based on KRT19 expression patterns

    • This allows for mapping of KRT19-high and KRT19-low cells within the same tumor microenvironment

    • Correlation with other markers can reveal previously unrecognized cellular states or phenotypes

  • Spatial Transcriptomics Integration:

    • Combining KRT19 expression data with spatial location information reveals organizational patterns within tumors

    • Understanding how KRT19-expressing cells interact with immune cells and stromal components in situ

    • Identification of regional differences in KRT19 expression that may correlate with invasive fronts or other tumor regions

  • Clonal Evolution Tracking:

    • Following how KRT19 expression changes during tumor progression and treatment

    • Identifying whether KRT19-expressing cells represent specific clonal populations with distinct genetic alterations

    • Determining if KRT19 expression changes precede or follow genetic or epigenetic alterations

  • Response Prediction:

    • Single-cell profiling before and after treatment to identify KRT19-related signatures predicting response

    • Characterization of residual disease following therapy based on KRT19 expression patterns

    • Development of more precise biomarkers based on single-cell KRT19 data rather than bulk tumor averages

  Studies utilizing CancerSEA database and other single-cell resources have begun exploring these approaches, suggesting that KRT19 expression at single-cell resolution may provide superior insights into tumor biology compared to traditional bulk tissue analysis .