KRT18 Monoclonal Antibody

What is KRT18 and what is its biological significance?

KRT18 (Cytokeratin 18) is a type I intermediate filament protein that forms heteropolymers with KRT8 and serves as an important structural component of epithelial cells. Biologically, KRT18 is involved in multiple cellular functions including:

• Uptake of thrombin-antithrombin complexes by hepatic cells

• Filament reorganization when phosphorylated

• Delivery of mutated CFTR to the plasma membrane

• Interleukin-6 (IL-6)-mediated barrier protection in conjunction with KRT8

The protein has several alternative names in scientific literature including CYK18, PIG46, Cell proliferation-inducing gene 46 protein, Cytokeratin-18, Keratin-18, CK-18, and K18 . As a key epithelial marker, KRT18 is widely used in research contexts for cell type identification and tissue characterization.

What applications are KRT18 monoclonal antibodies validated for?

KRT18 monoclonal antibodies have been validated for multiple research applications, with performance varying by specific clone. The primary validated applications include:

Researchers should perform antibody titration experiments to determine optimal concentrations for specific experimental conditions, as the recommended dilutions may vary based on sample type, fixation method, and detection system .

How should researchers evaluate the specificity of KRT18 monoclonal antibodies?

Evaluating antibody specificity is critical for ensuring reliable experimental results. For KRT18 monoclonal antibodies, comprehensive validation should include:

• Protein array testing: High-quality KRT18 antibodies undergo testing against extensive protein arrays (>19,000 full-length human proteins) to assess cross-reactivity with similar proteins. This is particularly important for KRT18 as it shares sequence similarity with other keratin family members .

• Western blot analysis: Verification that the antibody detects a protein of the expected molecular weight (approximately 45 kDa for KRT18) .

• Tissue panel reactivity: Testing across multiple tissue types with known KRT18 expression patterns. Positive staining should be observed in epithelial tissues while negative in non-epithelial tissues .

• Positive and negative cell line controls: Using cell lines with confirmed KRT18 expression (e.g., HeLa, MCF-7) versus those without expression .

• BLAST homology assessment: Computational prediction of potential cross-reactivity based on sequence homology with similar proteins, particularly other keratin family members .

For optimal experimental design, researchers should prioritize antibodies with documented evidence of mono-specificity, particularly those validated through protein arrays and multiple application methods .

How can KRT18 monoclonal antibodies be optimized for immunohistochemical applications?

Successful immunohistochemical detection of KRT18 requires careful optimization of multiple parameters:

• Epitope retrieval method: For formalin-fixed, paraffin-embedded tissues, heat-induced epitope retrieval (HIER) at pH 6.0 for 10-20 minutes followed by 20 minutes of cooling has been demonstrated effective . This step is critical as formalin fixation can mask KRT18 epitopes.

• Antibody concentration and incubation parameters:

  • For routine IHC: 1-3 μg/ml for 30 minutes at room temperature has shown optimal results

  • For overnight protocols: Lower concentrations (0.5-1 μg/ml) at 4°C may reduce background while maintaining specific signal

• Detection system selection: HRP polymer-based detection systems offer superior sensitivity compared to conventional ABC methods, with improved signal-to-noise ratios for KRT18 detection .

• Counterstain optimization: Nuclear counterstains should be optimized to provide contrast without obscuring cytoplasmic KRT18 signals. Hematoxylin dilution and incubation time may need adjustment based on tissue type .

• Tissue-specific considerations: Different epithelial tissues may require specific optimizations. For example, prostate carcinoma tissues have been successfully stained at 1 μg/ml, while colon tissues may require slight adjustments to antibody concentration for optimal results .

Importantly, researchers should always include appropriate positive controls (known KRT18-expressing tissues such as liver, colon, or prostate) and negative controls (antibody diluent only) in each IHC experiment to validate staining specificity .

What is the significance of KRT18 as a biomarker in cancer research?

KRT18 has emerged as an important biomarker in cancer research with multiple applications:

• Tumor classification and identification: As a marker of epithelial differentiation, KRT18 helps distinguish carcinomas from other cancer types such as sarcomas, lymphomas, and melanomas .

• Circulating tumor cell (CTC) detection: KRT18 antibodies are employed in multi-marker panels for identifying epithelial-derived CTCs in peripheral blood, which has prognostic and therapeutic monitoring applications . In combination with CD45 (PTPRC) antibodies (negative selection marker), KRT18 helps distinguish CTCs from leukocytes in liquid biopsy samples.

• Tumor progression assessment: Changes in KRT18 expression patterns correlate with tumor progression and aggressive behavior in multiple cancer types. Monitoring these changes can provide insights into disease progression .

• Apoptosis marker: During apoptosis, KRT18 undergoes specific caspase-mediated cleavage, generating fragments that can be detected with specific antibodies. This allows researchers to distinguish between different cell death mechanisms in tumor samples .

• Epithelial-mesenchymal transition (EMT) studies: Decreased KRT18 expression often accompanies EMT, a process associated with increased invasiveness and metastatic potential. Monitoring KRT18 alongside other EMT markers provides insights into this critical process .

Researchers investigating KRT18 in cancer contexts should consider using multiple epithelial markers (e.g., KRT8/KRT18 combinations) to improve specificity and sensitivity, particularly in poorly differentiated tumors where marker expression may be heterogeneous .

What technical considerations are important for multiplexed immunofluorescence with KRT18 antibodies?

Multiplexed immunofluorescence incorporating KRT18 detection requires careful technical considerations:

• Antibody panel design: When designing multiplexed panels:

  • Consider using KRT8/KRT18 combined antibodies for enhanced epithelial cell detection

  • Ensure species compatibility among primary antibodies to avoid cross-reactivity

  • Match fluorophores to available microscopy filter sets, considering spectral overlap

• Signal amplification strategies: For samples with low KRT18 expression:

  • Tyramide signal amplification (TSA) can enhance detection sensitivity

  • Quantum dot conjugates provide photostable alternatives to conventional fluorophores

  • Consider directly conjugated antibodies (e.g., FITC-conjugated KRT18 antibodies) to reduce background and simplify protocols

• Nuclear counterstaining optimization: DAPI counterstaining has been successfully used with KRT18 immunofluorescence but requires optimization:

  • Adjust DAPI concentration to avoid overwhelming KRT18 cytoplasmic signals

  • Consider alternative nuclear stains (RedDot) when using fluorophores with spectral overlap with DAPI

• Image acquisition parameters:

  • Sequential scanning reduces channel bleed-through for confocal microscopy

  • Z-stack imaging may be necessary to fully capture filamentous KRT18 structures

  • Exposure times should be optimized for each channel to prevent saturation

• Controls for multiplexed staining:

  • Single-stained controls for each marker are essential for spectral compensation

  • FMO (fluorescence minus one) controls help establish gating strategies for flow cytometry applications

An optimized example from the literature shows MCF-7 cells successfully stained with KRT8/KRT18-FITC labeled monoclonal antibody (green) and PTPRC-PE labeled monoclonal antibody (orange), with DAPI nuclear counterstain (blue) . This combination allows simultaneous visualization of epithelial tumor cells and exclusion of leukocytes in circulating tumor cell applications.

How does KRT18 interact with KRT8, and what are the research implications?

KRT18 and KRT8 form obligate heteropolymers that constitute the primary intermediate filament system in simple epithelial cells. This interaction has significant research implications:

Researchers should consider whether their scientific questions require specific detection of KRT18 alone or would benefit from simultaneous detection of both filament partners, particularly in applications studying epithelial integrity, stress responses, or epithelial-derived circulating tumor cells .

What are common challenges in KRT18 immunodetection and how can they be addressed?

Researchers working with KRT18 monoclonal antibodies may encounter several technical challenges:

• Variable epitope accessibility:

  • Problem: Formalin fixation can mask KRT18 epitopes, resulting in weak or absent staining.

  • Solution: Optimize epitope retrieval conditions. Boiling in citrate buffer (pH 6.0) for 10-20 minutes followed by 20 minutes cooling has proven effective . Alternative retrieval buffers (EDTA pH 9.0) may be tested if citrate buffer yields suboptimal results.

• Background staining:

  • Problem: Non-specific binding of antibodies to tissue components.

  • Solution: Implement additional blocking steps (protein block, avidin/biotin block for biotin-based detection systems), increase blocking duration, optimize antibody concentration through titration experiments, and consider adding mild detergents (0.1% Triton X-100 or Tween-20) to antibody diluents .

• Inconsistent staining patterns:

  • Problem: Variability in staining intensity or pattern between experiments.

  • Solution: Standardize fixation times, processing protocols, and staining conditions. Include positive control tissues (known KRT18-expressing epithelial tissues) in each experiment for quality control and comparison .

• Cross-reactivity with other keratins:

  • Problem: Some antibodies may cross-react with other keratin family members.

  • Solution: Select antibodies with documented specificity testing (e.g., protein array validation against >19,000 proteins) . Clone-specific validation data should be reviewed when selecting antibodies for critical applications.

• Signal detection sensitivity:

  • Problem: Weak signal in samples with low KRT18 expression.

  • Solution: Implement signal amplification methods such as tyramide signal amplification, polymer-based detection systems, or increase primary antibody incubation time (overnight at 4°C rather than 1 hour at room temperature) .

Each of these challenges requires systematic troubleshooting and optimization specific to the tissue type, fixation method, and detection system being employed.

How can researchers validate KRT18 antibody performance in their specific experimental systems?

Comprehensive validation of KRT18 antibodies in specific experimental systems should follow a multi-step approach:

• Positive and negative control tissues/cells:

  • Positive controls: Test antibodies on tissues with known KRT18 expression (liver, colon, prostate) or cell lines (HeLa, MCF-7)

  • Negative controls: Include tissues lacking epithelial cells (brain, skeletal muscle) or mesenchymal cell lines

  • Compare staining patterns with published literature and expected subcellular localization (cytoplasmic filamentous pattern)

• Antibody titration experiments:

  • Perform serial dilutions of antibody (e.g., 1:10, 1:50, 1:100, 1:500)

  • Determine optimal concentration that maximizes specific signal while minimizing background

  • Document titration results with standardized imaging parameters for future reference

• Specificity controls:

  • Perform peptide competition assays where available (pre-incubating antibody with immunizing peptide)

  • Compare results from multiple antibody clones targeting different KRT18 epitopes

  • Consider knockout/knockdown validation in cell lines when possible

• Western blot verification:

  • Confirm antibody detects a protein of expected molecular weight (45 kDa for KRT18)

  • Include positive control lysates from epithelial cells

  • Consider 2D Western blots to distinguish KRT18 from other proteins of similar molecular weight

• Orthogonal validation:

  • Correlate protein detection with mRNA expression data (RT-PCR or RNA-seq)

  • Compare results across multiple applications (IHC, IF, flow cytometry)

  • Document validation steps thoroughly for publication requirements

Following this structured validation approach provides confidence in antibody performance and experimental results, particularly for critical research applications or when developing novel methodologies .

What considerations are important when using KRT18 antibodies for flow cytometry?

Flow cytometric applications with KRT18 antibodies present unique challenges due to the intracellular localization of this protein:

• Fixation and permeabilization optimization:

  • Fixation: 4% paraformaldehyde (10-15 minutes) preserves cellular architecture while allowing antibody access

  • Permeabilization: Titrate permeabilization agents (0.1-0.5% saponin, 0.1-0.3% Triton X-100, or commercial permeabilization buffers) to optimize for specific antibody clones

  • Timing: Excessive permeabilization can damage epitopes while insufficient permeabilization prevents antibody access

• Antibody concentration and incubation parameters:

  • Flow cytometry typically requires higher antibody concentrations than other applications (1:20 dilution recommended for circulating tumor cell applications)

  • Extended incubation times (30-45 minutes) at room temperature improve signal intensity

  • Washing steps must be thorough but gentle to preserve cell integrity while removing unbound antibody

• Multiparameter panel design:

  • KRT18 detection is often combined with other markers in flow cytometry applications

  • Consider fluorophore brightness hierarchy (assign brightest fluorophores to lowest-expressed antigens)

  • Panel design should include:

    • Negative exclusion markers (e.g., CD45/PTPRC to exclude leukocytes)

    • Viability dyes to eliminate dead cells (which can bind antibodies non-specifically)

    • Additional epithelial markers for confirmation

• Gating strategy development:

  • Use isotype controls to set initial negative gates

  • Include fluorescence-minus-one (FMO) controls for accurate gate placement

  • Consider compensation controls when using multiple fluorophores with spectral overlap

• Sample-specific considerations:

  • For circulating tumor cell applications, specialized fixation protocols may be required

  • When analyzing mixed cell populations, additional markers should be included to ensure accurate identification of epithelial populations

  • Fresh samples typically provide better results than frozen/thawed cells

FITC-conjugated KRT8/KRT18 antibodies (e.g., clone CK8+18 207) have been successfully used in circulating tumor cell detection applications, with documented performance at 1:20 dilution in combination with PE-conjugated PTPRC antibodies and DAPI nuclear counterstain .

How is KRT18 utilized in circulating tumor cell research?

KRT18 antibodies have become essential tools in circulating tumor cell (CTC) research, with several methodological considerations:

• Detection principles and strategies:

  • KRT18 serves as a positive selection marker for epithelial-derived CTCs in peripheral blood

  • Combined with negative selection markers (CD45/PTPRC) to exclude leukocytes

  • Often incorporated into multiplexed panels with additional epithelial markers (EpCAM, other cytokeratins) for enhanced sensitivity

• Technical workflow optimization:

  • Sample preparation typically involves red blood cell lysis, Ficoll density gradient separation, or specialized CTC enrichment platforms

  • Fixation and permeabilization parameters require careful optimization to preserve rare CTCs while enabling intracellular antibody access

  • FITC-conjugated KRT8/KRT18 antibodies used at 1:20 dilution have demonstrated effective performance in CTC applications

• Immunofluorescence visualization:

  • CTCs are typically identified as KRT18+/CD45- nucleated cells

  • Fluorescence microscopy remains the gold standard for CTC confirmation

  • Documented protocols using MCF-7 cells demonstrate successful staining with KRT8/KRT18-FITC antibodies (green), PTPRC-PE antibodies (orange), and DAPI nuclear counterstain (blue)

• Quantitative analysis considerations:

  • Flow cytometry enables high-throughput analysis but requires careful gating strategies

  • Imaging flow cytometry combines morphological assessment with quantitative capability

  • Machine learning algorithms increasingly applied to automate CTC identification based on KRT18 staining patterns

• Clinical research applications:

  • CTCs detected using KRT18 antibodies serve as prognostic indicators in multiple cancer types

  • Serial monitoring of KRT18-positive CTCs may provide early indication of treatment response

  • Heterogeneity in KRT18 expression within CTCs may reflect tumor evolution and require consideration in research design

Researchers should be aware that epithelial-mesenchymal transition may downregulate KRT18 expression in some CTCs, potentially leading to false negatives. Combining KRT18 with mesenchymal markers provides a more comprehensive approach to CTC detection in advanced disease settings .

What are the emerging applications of KRT18 antibodies in cancer diagnostics research?

KRT18 antibodies are increasingly utilized in cancer diagnostics research beyond traditional applications, with several emerging areas:

• Liquid biopsy development:

  • Detection of KRT18 fragments in serum as biomarkers of epithelial cell death

  • Analysis of circulating KRT18-positive tumor cells using microfluidic and nanotechnology approaches

  • Development of automated, standardized detection systems for clinical translation

• Tumor heterogeneity assessment:

  • Spatial mapping of KRT18 expression within tumors using multiplexed immunofluorescence

  • Correlation of expression patterns with tumor subregions (invasive front vs. tumor core)

  • Integration with other markers to define functional tumor compartments

• Therapy response prediction:

  • Monitoring changes in KRT18 expression patterns following therapeutic intervention

  • Correlation of KRT18 fragment release with treatment efficacy

  • Development of companion diagnostic approaches for targeted therapies

• Metastasis research:

  • Analysis of KRT18 expression in metastatic lesions compared to primary tumors

  • Investigation of KRT18's role in metastatic colonization

  • Utilization as a marker for understanding the metastatic cascade

• Early detection strategies:

  • Evaluation of KRT18 as part of multimarker panels for early cancer detection

  • Application in screening high-risk populations

  • Incorporation into artificial intelligence-based diagnostic algorithms

These emerging applications leverage the specificity of KRT18 for epithelial cells and its differential expression patterns in malignancy, offering opportunities for enhanced diagnostic precision and personalized medicine approaches .

How does post-translational modification affect KRT18 detection and function?

Post-translational modifications (PTMs) of KRT18 significantly impact both its detection by antibodies and its biological functions:

• Phosphorylation effects:

  • KRT18 undergoes phosphorylation at multiple sites, particularly during mitosis and cellular stress

  • When phosphorylated, KRT18 plays important roles in filament reorganization

  • Phosphorylation can alter epitope accessibility, potentially affecting antibody binding

  • Phospho-specific KRT18 antibodies enable tracking of specific cellular states and stress responses

• Glycosylation considerations:

  • KRT18 contains O-GlcNAc modification sites that respond to cellular nutrient status

  • Glycosylation can mask antibody epitopes, potentially leading to false-negative results

  • Changes in glycosylation patterns occur during malignant transformation, affecting KRT18 detection

• Proteolytic processing:

  • During apoptosis, KRT18 undergoes specific caspase-mediated cleavage

  • Fragmented KRT18 serves as a biomarker for epithelial cell apoptosis

  • Antibodies targeting intact vs. cleaved forms enable discrimination between different cell death mechanisms

  • M30 antibody (specific for caspase-cleaved KRT18 fragment) is widely used in apoptosis research

• Acetylation impacts:

  • KRT18 undergoes acetylation that affects filament organization and solubility

  • Acetylation status changes in response to cellular stress

  • This modification may affect antibody binding depending on epitope location

• Methodological implications:

  • Sample preparation methods can preserve or destroy PTMs

  • Phosphatase inhibitors should be included when studying phosphorylated KRT18

  • Fixation methods differentially preserve PTMs (e.g., methanol better preserves phospho-epitopes than paraformaldehyde)

  • Antibody selection should consider the specific PTM status relevant to the research question

Researchers investigating specific cellular states or stress responses should consider these PTM effects and select antibodies appropriate for their research questions, potentially including modification-specific antibodies when studying particular cellular processes .

What novel approaches are emerging for KRT18 detection in research?

The field of KRT18 detection is evolving rapidly with several innovative approaches:

• Proximity ligation assays (PLA):

  • Enable detection of KRT18 interactions with binding partners

  • Provide enhanced specificity through dual antibody recognition

  • Allow visualization of protein-protein interactions in situ with single-molecule sensitivity

  • Particularly valuable for studying KRT18-KRT8 interactions and regulatory protein binding

• CRISPR-mediated endogenous tagging:

  • Enables live-cell imaging of KRT18 dynamics without antibodies

  • Fluorescent protein fusions or small epitope tags can be knocked into endogenous KRT18 loci

  • Preserves physiological expression levels and regulation

  • Eliminates concerns about antibody specificity and accessibility

• Aptamer-based detection:

  • DNA or RNA aptamers as alternatives to antibodies

  • Potentially higher specificity and reproducibility

  • Compatible with live-cell imaging applications

  • Less batch-to-batch variability compared to antibodies

• Mass cytometry (CyTOF):

  • Metal-conjugated antibodies enable highly multiplexed detection (>40 parameters)

  • No spectral overlap concerns as with fluorescence

  • Allows comprehensive phenotyping of KRT18-positive cells within heterogeneous populations

  • Enhanced ability to characterize rare subpopulations

• Single-cell sequencing integration:

  • Combining KRT18 protein detection with single-cell transcriptomics

  • CITE-seq and REAP-seq technologies link protein expression to transcriptional profiles

  • Enables correlation of KRT18 protein levels with global gene expression patterns

  • Provides insights into heterogeneity within KRT18-positive cell populations

These emerging approaches offer enhanced sensitivity, specificity, and contextual information compared to traditional antibody-based methods, potentially revealing new insights into KRT18 biology and function in various research contexts .

How can researchers effectively combine KRT18 detection with other molecular markers in complex experimental designs?

Designing effective multiplexed experiments incorporating KRT18 requires careful consideration of several factors:

• Complementary marker selection strategies:

  • Epithelial lineage confirmation: Combine KRT18 with EpCAM, E-cadherin, or additional cytokeratins

  • Differentiation status assessment: Pair with differentiation markers (CDX2, TTF1, PAX8) for tissue-of-origin studies

  • Functional state evaluation: Combine with proliferation markers (Ki67), apoptosis indicators (cleaved caspase-3), or EMT markers (vimentin, N-cadherin)

  • Microenvironmental context: Include immune cell markers, fibroblast markers, or vasculature indicators to understand tumor-stroma interactions

• Technical compatibility considerations:

  • Antibody species compatibility: Plan panels using primary antibodies from different host species to enable simultaneous detection

  • Sequential staining approaches: For same-species antibodies, implement sequential staining with blocking steps between rounds

  • Signal separation strategies: Utilize spectrally distinct fluorophores, subcellular localization differences (nuclear vs. cytoplasmic), or sequential bleaching approaches

• Advanced multiplexing technologies:

  • Cyclic immunofluorescence: Iterative staining and imaging allows >40 markers on a single sample

  • Mass cytometry (CyTOF): Metal-tagged antibodies enable high-parameter analysis without spectral overlap

  • Multiplexed ion beam imaging (MIBI): Enables spatial analysis of >40 proteins simultaneously

  • Digital spatial profiling (DSP): Combines imaging with quantitative protein measurement

• Data integration approaches:

  • Multi-omics correlation: Link KRT18 protein data with genomic or transcriptomic information

  • Spatial analysis: Perform neighborhood analysis to understand cellular interactions

  • Machine learning classification: Develop algorithms to identify complex cellular phenotypes based on marker combinations

  • Pseudotime trajectory analysis: Reconstruct differentiation or disease progression trajectories

• Validation strategies for multiplexed data:

  • Single-marker controls: Validate individual markers separately before combining

  • Known biological relationships: Confirm expected co-expression patterns (e.g., KRT8/KRT18)

  • Orthogonal validation: Verify key findings using alternative technologies

  • Biological replicates: Ensure reproducibility across multiple samples

By thoughtfully implementing these considerations, researchers can design robust multiplexed experiments that provide comprehensive insights into epithelial biology, tumor heterogeneity, and disease mechanisms while maintaining experimental rigor .