CRK14 Antibody

What is Cytokeratin 14 and what cellular structures does it localize to?

Cytokeratin 14 is a key component of the cytoskeleton in epithelial cells, providing structural support and resilience, particularly in the skin . At the subcellular level, CK14 localizes specifically to intermediate filaments within the cytoplasm . In normal human skin, CK14 expression is predominantly detected in keratinocytes . The protein functions as part of the epithelial cell scaffolding, contributing to cellular resilience and maintaining tissue architecture. When using fluorescent immunocytochemistry techniques, CK14 staining typically reveals a filamentous cytoplasmic pattern that highlights the intermediate filament network, as demonstrated in studies using normal human epidermal keratinocytes (NHEK) .

What are the primary research applications for CK14 antibodies?

CK14 antibodies are versatile tools employed across multiple experimental techniques. The primary validated applications include:

These applications enable researchers to investigate CK14 expression patterns across different experimental contexts, from protein quantification to spatial localization within cells and tissues .

Which cell types and tissues typically express CK14?

CK14 displays a distinctive expression pattern across human tissues:

• Epithelial tissues: Particularly abundant in the basal layer of stratified epithelia

• Skin: Strongly expressed in keratinocytes of the epidermis

• Breast tissue: Found in myoepithelial cells and a subpopulation of luminal cells in the terminal ductal lobular units (TDLUs)

• Cancer cell lines: Detected in certain carcinoma cell lines, such as A431 human epithelial carcinoma cells

Understanding this expression pattern is crucial for experimental design, as it provides context for interpreting results and selecting appropriate positive controls .

How is CK14 expression associated with specific breast cancer subtypes?

CK14 expression has emerged as a significant biomarker in breast cancer research, helping to identify specific molecular subtypes with distinct clinical behaviors:

• Basal-like breast cancers: Approximately 38% of breast carcinomas express stratified epithelial cytokeratins (including CK5, CK14, and/or CK17), which correlates with the basal-like molecular subtype

• Prognostic implications: Multiple studies have demonstrated correlations between poor prognosis and expression of CK5, CK14, and CK17

• Molecular associations: CK14-expressing tumors typically show negative expression of estrogen receptor (ER) and progesterone receptor (PR), forming a clinical subgroup distinct from c-erbB-2 expressing tumors

• Genomic correlations: Breast tumors expressing stratified epithelial keratins like CK14 display quantitative genomic abnormalities that distinguish them from tumors expressing only simple epithelial keratins

Interestingly, cytogenetic analyses have identified two subgroups of CK14-expressing tumors distinguished by different genomic alterations, suggesting further heterogeneity within this classification . These insights highlight the value of CK14 as both a diagnostic and prognostic marker in breast cancer research.

What is the relationship between CK14 and other cytokeratin markers in identifying cell lineages?

Research has revealed important co-expression patterns between CK14 and other cytokeratin markers:

• CK5/CK14 co-expression: Antibody KA1 recognizes CK5 in association with CK14, labeling a subpopulation of luminal cells in the terminal ductal lobular units (TDLUs) and basally located cells in the ducts of breast tissue

• Lineage tracing: CK14 expression patterns help distinguish epithelial from myoepithelial lineages in breast tissue, with myoepithelial lineages showing stable expression patterns with no signs of conversion

• Squamous differentiation: CK14 expression may be related to squamous metaplasia in certain contexts, as the "basal signature" also distinguishes squamous cells from adenocarcinoma in other tissues like lung cancer

This complex pattern of cytokeratin expression enables sophisticated lineage tracing experiments and helps identify cell populations with specific differentiation potentials. When designing experiments targeting specific epithelial subpopulations, researchers should consider using multiple cytokeratin markers to accurately identify cellular identities .

What are the current methodological approaches for validating CK14 antibody specificity?

Ensuring antibody specificity is critical for reliable research outcomes. Current best practices for validating CK14 antibody specificity include:

• Knockout validation: Confirming antibody specificity using knockout cell lines, as demonstrated with c-Kit antibody validation where signal was lost at the expected molecular weight in knockout cells but present in wild-type cells

• Multiple antibody comparison: Using multiple antibodies against the same target to confirm consistent staining patterns. Studies have shown that three different antibodies recognizing CK14 (LLO02, KA1, and EKH4) produced similar patterns of immunoreactivity with minor variations

• Cross-reactivity testing: Assessing potential cross-reactivity with other cytokeratin family members through careful controls and pre-absorption tests

• Tissue panel validation: Testing antibody performance across a panel of tissues with known CK14 expression patterns to confirm expected staining localization and intensity

What are the optimal sample preparation protocols for CK14 immunostaining?

Successful CK14 immunostaining requires careful attention to sample preparation:

For immunohistochemistry on paraffin-embedded sections:

• Fixation: Standard 10% neutral-buffered formalin fixation is generally suitable

• Antigen retrieval: Heat-mediated antigen retrieval using either:

  • Citrate buffer (pH 6.0) - assessed at 1:50 and 1:100 dilutions

  • Tris/EDTA buffer (pH 9.0) - demonstrated effective retrieval for related cytokeratin antibodies

• Enzymatic retrieval alternatives: Pepsin and pronase have been tested as alternative methods for CK14 antigen retrieval, though with limited success

For immunocytochemistry on cultured cells:

• Fixation method: Immersion fixation has been successfully employed for both A431 carcinoma cells and normal human epidermal keratinocytes (NHEK)

• Antibody concentration: 10 μg/mL applied for 3 hours at room temperature has yielded specific staining of cytoplasmic intermediate filaments

• Detection system: Secondary antibodies conjugated to fluorophores such as NorthernLights™ 557 have provided clear visualization of specific staining

These protocols should be optimized for specific experimental conditions, as variations in fixation time and tissue type may require adjustments to achieve optimal results .

What are the recommended dilutions and incubation conditions for different applications?

Optimal working conditions vary by application and specific antibody clone:

For specific examples:

• Mouse anti-human CK14 monoclonal antibody (MAB3164) demonstrated optimal staining at 10 μg/mL for 3 hours at room temperature for immunofluorescence applications

• For IHC on paraffin sections, 3 μg/mL applied for 1 hour at room temperature followed by anti-mouse IgG HRP polymer detection has yielded specific staining of keratinocytes in human skin samples

These conditions should serve as starting points, with optimization recommended for each specific experimental system .

How can multiplex staining with CK14 and other markers be effectively implemented?

Multiplex staining enables simultaneous visualization of multiple markers within the same sample:

• Compatible markers: CK14 antibodies have been successfully combined with other markers such as CKAP4/p63 in co-staining protocols

• Secondary antibody selection: When performing multiplex staining, careful selection of species-specific secondary antibodies with distinct fluorophores is essential to avoid cross-reactivity

  • Example: Mouse anti-human CK14 detected with NorthernLights™ 557-conjugated anti-mouse IgG (red) combined with Sheep anti-human CKAP4/p63 detected with NorthernLights™ 493-conjugated anti-sheep IgG (green)

• Nuclear counterstaining: DAPI (blue) provides effective nuclear counterstaining that doesn't interfere with cytoplasmic CK14 staining

• Sequential vs. simultaneous: For some combinations, sequential staining may be preferable to simultaneous incubation to minimize cross-reactivity

When designing multiplex experiments, researchers should first validate each antibody individually before attempting co-staining to ensure specific signal detection and proper controls .

What are common technical challenges when working with CK14 antibodies and how can they be addressed?

Researchers may encounter several technical issues when working with CK14 antibodies:

One specific challenge noted in the literature is that some anti-keratin 14 antibodies are not specific to the basal layer of the skin and may target all epithelial layers . Researchers should be aware of this potential limitation when interpreting results related to skin tissue stratification .

How should researchers interpret CK14 expression patterns in the context of cancer research?

Interpreting CK14 expression in cancer research requires consideration of several factors:

• Tumor heterogeneity: CK14 expression may not be uniform throughout a tumor sample, necessitating analysis of multiple regions to establish representative expression patterns

• Correlation with clinical parameters: CK14 expression should be interpreted alongside other biomarkers and clinical data, as its prognostic significance varies by cancer type

• Quantitative assessment: Consider both staining intensity and percentage of positive cells when evaluating CK14 expression

  • Some studies employ hierarchical cluster analysis to identify clinical subgroups based on CK14 expression alongside other proteins

• Molecular context: CK14-positive tumors often show specific molecular associations, including:

  • Negative correlation with hormone receptor expression (ER/PR negative)

  • Positive correlation with p53 expression

  • Increased expression of epidermal growth factor receptor (EGFR)

  • Higher proliferative index

These correlations can help researchers stratify tumors into molecular subtypes with distinct biological behaviors and therapeutic vulnerabilities. For instance, the association between CK14 expression and certain medullary carcinomas (both typical and atypical) provides important diagnostic and prognostic information .

What quality control measures are essential for ensuring reproducible CK14 antibody-based experiments?

Ensuring experimental reproducibility requires rigorous quality control:

• Antibody validation: Confirm antibody specificity through techniques like Western blotting against known positive and negative cell lines or tissues

• Lot-to-lot consistency: Test new antibody lots against previous lots to ensure consistent performance

• Positive and negative controls: Include appropriate controls in each experiment:

  • Positive tissue controls (e.g., human skin for CK14)

  • Negative controls (omitting primary antibody)

  • Isotype controls to assess non-specific binding

• Standardized protocols: Document detailed protocols including:

  • Antibody dilution and incubation time

  • Antigen retrieval method and duration

  • Detection system specifications

  • Image acquisition parameters

• Equilibrium dissociation constant (KD) assessment: For recombinant antibodies, KD values provide objective measures of binding affinity, with recombinant antibodies typically showing 1-2 orders of magnitude higher affinity than traditional mouse monoclonals

Implementing these quality control measures increases confidence in experimental results and facilitates reproducibility across different research groups and experiments .

How are CK14 antibodies contributing to our understanding of epithelial cell biology and disease mechanisms?

CK14 antibodies continue to advance our understanding of fundamental biological processes and disease mechanisms:

• Lineage tracing: CK14 antibodies enable tracking of epithelial cell populations during development, homeostasis, and disease progression

• Cancer heterogeneity: Studies using CK14 antibodies have revealed distinct molecular subtypes of breast cancer with different prognostic outcomes

• Epithelial-mesenchymal transition (EMT): Monitoring changes in CK14 expression during EMT provides insights into cancer progression mechanisms

• Diagnostic applications: CK14 expression patterns help distinguish between different types of carcinomas and inform treatment decisions

Future research will likely focus on integrating CK14 expression data with other molecular markers to develop more precise diagnostic and prognostic tools, particularly in the context of precision oncology approaches .

What emerging technologies are enhancing the application of CK14 antibodies in research?

Several technological advances are expanding the utility of CK14 antibodies:

• Single-cell analysis: Integration of CK14 immunostaining with single-cell RNA sequencing to correlate protein expression with transcriptional profiles

• Spatial transcriptomics: Combining CK14 immunohistochemistry with spatial gene expression analysis to map cellular heterogeneity within tissues

• Live-cell imaging: Development of non-toxic fluorescently-tagged antibody fragments for tracking CK14 dynamics in living cells

• Mass cytometry (CyTOF): Incorporation of CK14 antibodies into multi-parameter panels for simultaneous detection of dozens of proteins at the single-cell level

• Automated image analysis: AI-based quantification of CK14 staining patterns for objective assessment of expression levels and subcellular localization

These technological developments are enabling more sophisticated experimental approaches and generating richer datasets that reveal the complexities of CK14 biology in normal and pathological contexts .