KRT18 Antibody

What is KRT18 and where is it typically expressed?

KRT18 (Cytokeratin 18) is a type I intermediate filament protein with a molecular weight of approximately 45 kDa that forms part of the cytoskeletal structure in epithelial cells . It is primarily expressed in single layer epithelial tissues throughout the body, rather than in stratified squamous epithelia. KRT18 is found in tissues from the gastrointestinal tract, respiratory tract, urogenital tract, as well as endocrine and exocrine tissues and mesothelial cells . Notably, KRT18 typically exists in combination with cytokeratin 8 (KRT8) as a heterodimeric partner, which contributes to its structural integrity and functional properties within the cytoskeleton . Its cytoplasmic localization makes it an excellent marker for identifying cells of epithelial origin in both normal and pathological tissue samples.

What are the key biological functions of KRT18 beyond structural support?

KRT18 performs several critical cellular functions that extend beyond its structural role:

• Participates in the uptake of thrombin-antithrombin complexes by hepatic cells

• When phosphorylated, plays a crucial role in filament reorganization within the cell cytoskeleton

• Involved in the delivery of mutated CFTR (Cystic Fibrosis Transmembrane Conductance Regulator) to the plasma membrane, suggesting a role in protein trafficking

• Works together with KRT8 in interleukin-6 (IL-6)-mediated barrier protection, indicating involvement in immune response regulation

• Contributes to cellular processes related to migration and proliferation, particularly in cancer contexts, as demonstrated by knockdown experiments showing reduced proliferation and migration in lung cancer cell lines

Understanding these diverse functions helps explain why KRT18 dysregulation may contribute to various pathological conditions beyond simply serving as a diagnostic marker.

What applications are most suitable for KRT18 antibodies in research?

KRT18 antibodies have been validated for multiple laboratory applications with specific performance characteristics:

The most appropriate application depends on your specific research question. For tissue-based studies, IHC-P is particularly effective, while cell-based studies may benefit from ICC or flow cytometry approaches depending on whether morphological information or quantitative analysis is prioritized .

How should researchers optimize immunohistochemical protocols for KRT18 detection?

For optimal immunohistochemical detection of KRT18 in tissue samples:

• Tissue preparation: Formalin-fixed, paraffin-embedded (FFPE) tissues at 4-6 μm thickness provide excellent results, as demonstrated in validation studies using human prostate carcinoma and rat colon tissues .

• Antigen retrieval: Heat-induced epitope retrieval (HIER) in citrate buffer (pH 6.0) is typically most effective for unmasking KRT18 epitopes that may be cross-linked during fixation.

• Blocking and antibody concentration: Apply KRT18 antibody at optimized concentration, typically around 1 μg/ml for most validated antibodies . Use serum-free protein block to minimize background staining.

• Detection system: Employ a polymer-based detection system compatible with the primary antibody host species (mouse monoclonal or rabbit recombinant, depending on the clone) .

• Positive controls: Include known KRT18-expressing tissues such as liver, colon, or pancreas as positive controls in each staining batch.

The specificity of different clones varies; for example, KRT18/2808R (rabbit monoclonal) reacts with a wide variety of simple epithelia but not with stratified squamous epithelia, and specifically recognizes epithelial tumors of the gastrointestinal tract, lung, breast, pancreas, ovary, and thyroid .

What considerations are important when selecting between different KRT18 antibody clones?

When selecting a KRT18 antibody clone, researchers should consider:

• Epitope specificity: Different clones (such as KRT18/1190, KRT18/836, KRT18/2808R) recognize distinct epitopes on the KRT18 protein, which may affect detection in certain experimental contexts .

• Host species: Available options include mouse monoclonal (like KRT18/1190) and rabbit recombinant monoclonal (like KRT18/2808R) antibodies, which may influence compatibility with other antibodies in multiplex experiments .

• Species reactivity: Some clones are reactive with human KRT18 only, while others cross-react with rat or other species, important for translational research .

• Application validation: Not all clones perform equally across applications. For example, some may excel in IHC-P but perform poorly in flow cytometry. Review validation data for your specific application .

• Conjugation options: Consider whether native (unconjugated) or fluorophore-conjugated antibodies (such as Alexa Fluor® 700 or Alexa Fluor® 750) better suit your experimental design, particularly for multiplex applications .

For most comprehensive studies, it's advisable to validate at least two different clones to confirm findings and rule out clone-specific artifacts.

How can KRT18 antibodies be effectively used in cancer research?

KRT18 antibodies offer several valuable applications in cancer research:

• Tumor classification: KRT18 antibodies help distinguish adenocarcinomas (typically KRT18-positive) from squamous cell carcinomas and other non-epithelial malignancies, aiding in precise tumor classification and diagnosis .

• Functional studies: As demonstrated in lung adenocarcinoma research, KRT18 knockdown experiments followed by antibody-based validation have revealed its critical role in cancer cell proliferation and migration . Specifically, siRNA-mediated KRT18 knockdown in A549 and H1299 lung cancer cell lines showed significantly reduced proliferation in CCK-8 assays and diminished migratory capacity in scratch assays .

• Mechanistic investigations: KRT18 antibodies can be used to study interactions with other proteins or signaling pathways to elucidate its role in cancer progression and metastasis.

• Therapy response monitoring: Changes in KRT18 expression or fragmentation patterns following treatment may serve as indicators of therapeutic efficacy.

• Metastatic disease tracking: KRT18 antibodies can help identify cells of epithelial origin in circulation or distant sites, potentially aiding in metastasis research.

The emerging role of KRT18 in lung adenocarcinoma development, as validated through integrative bioinformatics and experimental approaches, highlights the importance of this marker beyond its traditional diagnostic applications .

What are the methodological considerations for studying KRT18 in relation to cell migration and proliferation?

When investigating KRT18's role in cell migration and proliferation, researchers should consider:

• Knockdown validation: When using siRNA or shRNA to suppress KRT18 expression, it's essential to confirm knockdown efficiency at both mRNA level (using q-PCR) and protein level (using Western blot with KRT18 antibodies) .

• Proliferation assays: The CCK-8 assay has been successfully used to demonstrate reduced proliferation following KRT18 knockdown in lung cancer cells . Alternative methods include BrdU incorporation, Ki-67 staining, or real-time cell analysis systems.

• Migration assessment: Scratch wound healing assays effectively demonstrate KRT18's impact on cell migration capability . Consider supplementing with transwell migration assays or time-lapse microscopy for more detailed analysis.

• Control selection: Proper controls are critical - use non-targeting siRNA sequences with similar GC content for knockdown experiments, and include appropriate isotype controls for antibody-based detection.

• Temporal considerations: Monitor changes over multiple time points, as demonstrated in the lung cancer studies where proliferation and migration were tracked at 24, 48, and 72 hours post-knockdown .

• Cell line selection: Consider testing multiple cell lines to ensure findings are not cell line-specific; the lung cancer studies validated findings in both A549 and H1299 cell lines .

These methodological approaches provide robust frameworks for investigating KRT18's functional roles in cancer and other biological contexts.

How can KRT18 antibodies be integrated into multiplex immunofluorescence protocols?

For effective multiplex immunofluorescence incorporating KRT18 antibodies:

• Antibody selection: Choose fluorophore-conjugated KRT18 antibodies (such as Alexa Fluor® 700 or Alexa Fluor® 750) for direct detection, or use unconjugated primary antibodies with spectrally distinct secondary antibodies .

• Panel design: Consider combining KRT18 with:

  • KRT8 (its heterodimeric partner) to assess co-expression patterns

  • Proliferation markers (Ki-67) when studying growth dynamics

  • EMT markers (E-cadherin, vimentin) to investigate epithelial-mesenchymal transition

  • Tissue-specific markers for contextual analysis

• Spectral considerations: When using multiple fluorophores, ensure minimal spectral overlap between channels. Far-red fluorophores like Alexa Fluor® 750 paired with KRT18 antibodies can help minimize bleed-through in multicolor experiments .

• Sequential staining approach: For complex panels, consider sequential rather than simultaneous staining, with intermediate fixation steps to prevent cross-reactivity between antibodies.

• Validation controls: Include single-stained controls for each marker to facilitate compensation during image analysis and confirm staining specificity.

• Image acquisition parameters: Optimize exposure times for each channel independently to balance signal intensity while avoiding oversaturation.

Properly designed multiplex panels can provide rich contextual information about KRT18 expression in relation to other markers, enhancing understanding of its biological significance.

What are common issues encountered with KRT18 antibody staining and how can they be resolved?

Researchers frequently encounter these challenges when working with KRT18 antibodies:

When troubleshooting KRT18 staining issues, systematically modify one variable at a time, beginning with antigen retrieval optimization, followed by antibody concentration adjustment, and finally protocol modifications for specific tissues or applications.

How should KRT18 antibody specificity be validated in experimental systems?

Rigorous validation of KRT18 antibody specificity should include:

• Positive and negative tissue controls: Confirm staining in tissues known to express KRT18 (simple epithelia) and absence of staining in negative tissues (lymphoid tissues, stratified squamous epithelia) .

• Genetic validation approaches:

  • siRNA knockdown experiments as demonstrated in lung cancer cell lines, where KRT18 suppression resulted in correspondingly decreased antibody signal

  • CRISPR/Cas9 knockout models where available

  • Overexpression systems with tagged KRT18 constructs

• Western blot validation: Confirm detection of a single band at the expected molecular weight (45 kDa), as this helps distinguish KRT18 from other cytokeratins of similar molecular weights .

• Comparison across antibody clones: Test multiple KRT18 antibody clones targeting different epitopes (e.g., KRT18/1190 vs. KRT18/2808R) to confirm consistent staining patterns .

• Orthogonal method validation: Correlate protein detection with mRNA expression using RT-PCR or RNA sequencing data .

Thorough validation is particularly important when applying KRT18 antibodies to novel tissue types, disease states, or experimental conditions where expression patterns may differ from established paradigms.

What factors influence KRT18 expression that might affect antibody detection?

Several biological and experimental factors can influence KRT18 expression and detection:

• Tissue differentiation state: KRT18 expression varies with epithelial differentiation, potentially leading to heterogeneous staining within a single tissue .

• Pathological conditions: Disease states, particularly malignant transformation, can alter KRT18 expression levels and patterns, requiring optimization of detection protocols .

• Epithelial-mesenchymal transition (EMT): During EMT, epithelial markers including KRT18 may be downregulated, potentially leading to false-negative results in cells undergoing this transition.

• Post-translational modifications: Phosphorylation states of KRT18 can affect filament organization and potentially antibody epitope accessibility .

• Fixation and processing artifacts: Overfixation can mask epitopes, while insufficient fixation may lead to antigen loss; optimize fixation protocols for each tissue type.

• Species differences: Sequence variations between species may affect antibody cross-reactivity; for example, some KRT18 antibodies react with human but not canine samples .

Understanding these factors helps researchers interpret KRT18 staining patterns correctly and design appropriate controls to account for potential variability in detection.

How is KRT18 being utilized in lung cancer research beyond diagnostic applications?

Recent advances in lung cancer research have expanded KRT18's utility beyond diagnosis:

• Functional role assessment: Experimental knockdown of KRT18 in lung cancer cell lines (A549 and H1299) demonstrates its critical role in maintaining proliferative and migratory capabilities . Specifically, siRNA-mediated KRT18 suppression significantly reduced cell proliferation as measured by CCK-8 assay and impaired migration in scratch wound healing assays .

• Integrative analysis approach: Recent research combines bioinformatics-based gene expression analysis with experimental validation to elucidate KRT18's role in lung adenocarcinoma development, representing a powerful methodological paradigm .

• Prognostic implications: Emerging evidence suggests KRT18 expression patterns may correlate with clinical outcomes in lung cancer patients, potentially serving as prognostic biomarkers.

• Mechanistic insights: Ongoing research is investigating the molecular pathways through which KRT18 influences cancer cell behavior, including potential interactions with signaling networks involved in proliferation and migration.

• Therapeutic targeting considerations: The demonstrated effects of KRT18 knockdown on reducing cancer cell proliferation and migration suggest potential therapeutic relevance, either as a direct target or as a biomarker for response to specific treatments .

These multifaceted approaches highlight KRT18's emerging significance beyond its traditional role as a diagnostic marker in lung cancer research.

What methodological advances are improving KRT18 detection sensitivity and specificity?

Recent technical innovations enhancing KRT18 detection include:

• Recombinant monoclonal antibody technology: Newer recombinant rabbit monoclonal antibodies (such as KRT18/2808R) offer improved lot-to-lot consistency and specificity compared to traditional hybridoma-derived antibodies .

• Conjugated antibody formats: Direct conjugation with far-red fluorophores (e.g., Alexa Fluor® 700, Alexa Fluor® 750) enables more sensitive detection with minimal autofluorescence interference and facilitates multiplex applications .

• Epitope-specific antibodies: Development of antibodies targeting specific forms of KRT18 (e.g., phosphorylated or cleaved variants) allows for more nuanced analysis of KRT18 biology.

• Signal amplification systems: Polymer-based detection systems and tyramide signal amplification protocols enhance sensitivity for detecting low-level KRT18 expression.

• Automated image analysis: Machine learning-based image analysis algorithms improve quantification of KRT18 expression patterns and subcellular localization.

• Combined protein-mRNA detection: Protocols integrating KRT18 protein detection with mRNA visualization (such as RNAscope with immunofluorescence) provide comprehensive expression analysis at both transcriptional and translational levels.

These methodological advances collectively enhance researchers' ability to detect and analyze KRT18 with greater precision in diverse experimental contexts.

How does KRT18 interact with other cytokeratins, and what implications does this have for antibody-based studies?

Understanding KRT18's interactions with other cytokeratins has important implications for experimental design:

• Obligate heterodimerization: KRT18 predominantly forms heterodimers with KRT8 in simple epithelia; this pairing is essential for proper intermediate filament formation . Consequently, co-expression analysis of both partners provides more complete information than studying either in isolation.

• Tissue-specific expression patterns: While KRT18 is widely expressed in simple epithelia, it shows tissue-specific co-expression patterns with other cytokeratins. For example, gastrointestinal tissues express both KRT8 and KRT18 but typically lack KRT14 .

• Compensatory regulation: Knockdown or mutation of one keratin partner may affect the expression, stability, or localization of the other, complicating interpretation of single-keratin studies.

• Filament reorganization dynamics: Phosphorylation of KRT18 affects filament reorganization, potentially altering interactions with KRT8 and other cytoskeletal components . Studies of KRT18 should consider these dynamic modifications.

• Cross-reactivity considerations: Due to structural similarities among cytokeratins, antibody cross-reactivity is a potential concern. Thorough validation is essential to ensure KRT18 specificity, particularly in tissues expressing multiple cytokeratin types.

• Functional cooperation: KRT18 works together with KRT8 in processes such as IL-6-mediated barrier protection , suggesting that functional studies should consider both partners rather than KRT18 alone.

These complex interactions highlight the importance of a comprehensive approach when designing experiments to study KRT18 in epithelial biology and pathology.