KRT14 Monoclonal Antibody

What is KRT14 and what is its biological significance?

Keratin 14 (KRT14) is a 50 kDa type I acidic keratin protein encoded by the KRT14 gene located at chromosome 17q21.2. It functions as a critical structural component of the cytoskeletal scaffold within epithelial cells, contributing to cellular architecture and providing mechanical resilience. KRT14 typically forms heterodimers with type II keratin 5 (KRT5) to create intermediate filaments that anchor the epidermis to underlying skin layers and attach keratinocytes together. This structural network is essential for maintaining skin integrity and protecting against everyday physical stress. The importance of KRT14 is underscored by the fact that mutations in the KRT14 gene are associated with skin disorders such as epidermolysis bullosa simplex and dermatopathia pigmentosa reticularis, which are characterized by skin fragility and blistering .

KRT14 expression is predominantly found in basal cells of stratified epithelia, including the skin and non-keratinizing squamous epithelium. It is also expressed in sebaceous glands, hair follicles, thymic epithelial cells including Hassall's corpuscles, tonsil crypt epithelium, basal cells of the prostate and respiratory epithelium, and in myoepithelial cells of various glandular tissues . This specific expression pattern makes KRT14 a valuable marker for identifying cell types and structures in basic and clinical research.

What are the primary applications of KRT14 monoclonal antibodies in research?

KRT14 monoclonal antibodies serve multiple critical functions in biomedical research:

• Western Blotting (WB): Enables quantitative assessment of KRT14 protein expression in tissue and cell lysates, typically visualized at approximately 50 kDa .

• Immunohistochemistry (IHC): Allows visualization of KRT14 expression patterns in tissue sections, crucial for studying tissue architecture and cellular differentiation states .

• Flow Cytometry (FACS): Facilitates identification and isolation of KRT14-expressing cell populations, particularly valuable in stem cell and cancer research .

• Immunocytochemistry (ICC): Enables visualization of KRT14 in cultured cells to study cytoskeletal organization and epithelial cell biology .

• Tumor Classification: Aids in identifying and classifying epithelial tumors, particularly basal-like breast cancers and squamous cell carcinomas .

• Developmental Biology: Helps track epithelial lineage specification during tissue development .

• Disease Mechanism Studies: Facilitates investigation of pathological mechanisms in KRT14-related disorders .

The selection of application determines the optimal clone, host species, and conjugation status of the antibody, with different research questions requiring specific antibody characteristics.

How should researchers select the appropriate KRT14 monoclonal antibody for their experiments?

Selection of the appropriate KRT14 monoclonal antibody requires careful consideration of multiple parameters:

When selecting between multiple options, prioritize antibodies with validation in your specific application and experimental system. Review literature to identify antibody clones commonly used in your field to facilitate comparison with published results.

What controls should be implemented when working with KRT14 antibodies?

Implementing appropriate controls is critical for ensuring experimental validity and interpretable results:

Positive Controls:

• Tonsil tissue sections should show strong KRT14 immunostaining in surface and crypt epithelium .

• Skin sections should demonstrate strong staining in basal keratinocytes.

• Cell lines known to express KRT14 (e.g., certain breast or squamous cell carcinoma lines).

Negative Controls:

• Tonsil sections should show absence of KRT14 immunostaining in all non-epithelial cells .

• Tissues known to lack KRT14 expression (e.g., lung, liver, pancreas).

• Primary antibody omission control to assess non-specific binding of detection systems.

• Isotype control matched to the primary antibody's host species and isotype.

Technical Controls:

• Blocking peptide competition assay to confirm antibody specificity.

• If using multiple detection methods, include single-stained controls.

• Include unstained cells/tissues for autofluorescence assessment in fluorescence applications.

• Consider siRNA knockdown or KRT14-null cell lines as specificity controls.

Consistent use of these controls will enhance data reliability and facilitate troubleshooting if unexpected results occur.

How can KRT14 expression patterns be utilized to distinguish different epithelial tumor types?

KRT14 expression analysis offers valuable diagnostic and prognostic information in tumor classification:

Breast Cancer:

• KRT14 is a key marker of basal-like breast cancer subtypes, which typically show more aggressive clinical behavior .

• Co-expression with KRT5/6 strengthens identification of basal-like phenotypes.

• The presence of KRT14-positive cells at the invasive front of tumors may indicate increased invasive potential.

Squamous Cell Carcinomas (SCCs):

• KRT14 expression is characteristic of SCCs arising from various anatomical sites, including skin, head and neck, lung, esophagus, and cervix .

• The pattern and intensity of KRT14 staining can help distinguish well-differentiated from poorly differentiated SCCs.

Salivary Gland Tumors:

• KRT14 positivity in myoepithelial components can aid in differentiating tumor types with myoepithelial participation.

• The distribution pattern of KRT14-positive cells helps distinguish adenoid cystic carcinomas from polymorphous adenocarcinomas.

Prostate Cancer:

• The presence of KRT14-positive basal cells helps distinguish benign prostatic hyperplasia from prostatic adenocarcinoma (which typically lacks KRT14-positive basal cells).

• The emergence of KRT14-positive cells in prostate cancer may indicate treatment resistance or neuroendocrine differentiation.

When implementing KRT14 immunostaining for tumor classification, it is advisable to use it within a panel of markers rather than as a standalone diagnostic tool. The interpretation of KRT14 expression patterns requires consideration of staining intensity, distribution, and correlation with morphological features and clinical parameters.

What are the methodological considerations for optimizing KRT14 detection in formalin-fixed paraffin-embedded tissues?

Optimizing KRT14 detection in formalin-fixed paraffin-embedded (FFPE) tissues requires addressing several methodological challenges:

Antigen Retrieval Optimization:

• Heat-induced epitope retrieval (HIER) is generally preferred for KRT14 detection.

• Compare citrate buffer (pH 6.0) versus EDTA buffer (pH 9.0) to determine optimal retrieval conditions.

• Adjust retrieval duration (10-30 minutes) based on tissue type and fixation conditions.

Antibody Dilution and Incubation:

• Start with manufacturer-recommended dilutions (typically 1:50-1:100 for IHC) .

• Perform titration experiments to determine optimal signal-to-noise ratio.

• Consider whether room temperature (1-2 hours) or overnight incubation (4°C) provides superior results.

Detection System Selection:

• For routine IHC, avidin-biotin complex (ABC) systems are effective .

• Polymer-based detection systems may offer improved sensitivity with less background.

• Tyramide signal amplification can enhance detection of low-abundance KRT14.

Background Reduction Strategies:

• Include additional blocking steps with normal serum matched to the host of the secondary antibody.

• Consider low-protein blocking solutions to reduce non-specific binding.

• Implement stringent washing protocols between incubation steps.

Counterstaining Considerations:

• Use light hematoxylin counterstaining to avoid masking KRT14 signal in basal cells.

• Ensure dehydration steps do not extract the chromogen.

A systematic approach to optimization, including side-by-side comparison of methods and documentation of results, will yield the most consistent and reproducible KRT14 detection protocol for your specific tissue types and research questions.

How can researchers troubleshoot inconsistent KRT14 staining in experimental procedures?

Troubleshooting inconsistent KRT14 staining requires systematic analysis of potential technical and biological variables:

Technical Variables:

Biological Variables:

• Tissue Heterogeneity: KRT14 expression can vary within the same tissue. Sample multiple regions to assess spatial heterogeneity.

• Fixation Effects: Overfixation can mask epitopes. Document fixation conditions and consider this variable when comparing samples.

• Differentiation State: KRT14 expression changes with epithelial differentiation. Consider cell differentiation status when interpreting results.

• Species Differences: If working across species, verify antibody cross-reactivity with positive control tissues from each species.

• Disease State Impact: Pathological conditions may alter protein expression or accessibility. Compare with appropriate disease-matched controls.

Maintaining a detailed laboratory notebook documenting all procedural variables (reagent lots, incubation times/temperatures, sample processing) facilitates identification of sources of variability. When troubleshooting, change only one variable at a time and include appropriate controls with each experiment.

What are the considerations for using KRT14 monoclonal antibodies in multiplex immunofluorescence assays?

Implementing KRT14 detection in multiplex immunofluorescence requires careful attention to several technical considerations:

Antibody Panel Design:

• Select KRT14 antibodies validated specifically for immunofluorescence applications .

• Consider the host species of all panel antibodies to avoid cross-reactivity issues.

• Verify that KRT14 antibody concentration needs are compatible with your multiplex protocol.

• Test single-stain controls for each antibody before combining them.

Spectral Considerations:

• Choose fluorophores with minimal spectral overlap for KRT14 and other targets.

• If using direct conjugates, select a bright fluorophore for KRT14 if it's expressed at low levels.

• Consider the autofluorescence characteristics of your tissue type when selecting fluorophores.

• Implement appropriate spectral unmixing if using spectrally adjacent fluorophores.

Sequential Staining Considerations:

• Determine if the KRT14 epitope is sensitive to harsh elution conditions if planning sequential rounds.

• Consider whether KRT14 should be detected in earlier or later rounds based on abundance.

• Validate that signal intensity remains consistent across staining rounds.

Image Analysis Optimization:

• Establish threshold values for KRT14 positivity based on positive and negative controls.

• Consider subcellular localization patterns (cytoplasmic for KRT14) when designing segmentation algorithms.

• Implement quality control metrics to identify and exclude artifacts.

Validation Approaches:

• Compare multiplex results with single-plex staining on serial sections.

• Include samples with known KRT14 expression patterns as technical controls.

• Consider orthogonal validation with RNA expression data where possible.

For quantitative analysis of KRT14 in multiplex assays, standardization of image acquisition parameters and analysis workflows is critical for generating reproducible results across experiments and between laboratories.

How can researchers correlate KRT14 expression with clinical outcomes in cancer research studies?

Correlating KRT14 expression with clinical outcomes requires rigorous methodological approaches:

Cohort Selection and Characterization:

• Define clear inclusion/exclusion criteria for patient samples.

• Collect comprehensive clinical data including treatment history, response, and outcomes.

• Consider potential confounding variables (age, stage, grade, treatment regimens).

• Calculate appropriate sample sizes based on expected effect sizes and desired statistical power.

KRT14 Assessment Methods:

• Develop standardized scoring systems for KRT14 immunohistochemistry:

  • Percentage of positive cells (0-100%)

  • Staining intensity (0-3+)

  • H-score calculation (percentage × intensity)

  • Consider both average and focal expression patterns

• For multiplexed approaches, quantify:

  • KRT14 colocalization with other markers

  • Spatial relationships between KRT14+ cells and other tissue components

  • KRT14+ cell density in different tumor regions

Statistical Analysis Approaches:

Integration with Other Biomarkers:

• Assess KRT14 in the context of established biomarkers for the specific cancer type.

• Consider creating composite scores incorporating KRT14 with complementary markers.

• Explore potential biological interactions between KRT14 and other markers.

When publishing KRT14 correlation studies, report detailed methodological information including antibody clone, dilution, scoring criteria, and statistical approaches to facilitate reproducibility and comparison across studies. Additionally, consider the functional significance of KRT14 expression in your cancer type based on its known biological roles in cell structure, migration, and epithelial differentiation.

What is the recommended protocol for KRT14 western blotting?

The following protocol outlines optimized conditions for detecting KRT14 in western blotting applications:

Sample Preparation:

• Extract total protein from tissues or cells using RIPA buffer supplemented with protease inhibitors.

• Determine protein concentration using BCA or Bradford assay.

• Prepare samples at 1-2 μg/μL in Laemmli buffer with reducing agent.

• Heat samples at 95°C for 5 minutes.

Gel Electrophoresis and Transfer:

• Load 10-20 μg of protein per lane on a 10-12% SDS-PAGE gel.

• Include molecular weight markers covering the 50 kDa range.

• Run gel at 100-120V until the dye front reaches the bottom.

• Transfer to PVDF membrane at 100V for 1 hour or 30V overnight at 4°C.

Antibody Incubation:

• Block membrane with 5% non-fat dry milk or BSA in TBST for 1 hour at room temperature.

• Incubate with KRT14 primary antibody at 1:1000 dilution in blocking buffer overnight at 4°C.

• Wash 3 times with TBST, 5 minutes each.

• Incubate with appropriate HRP-conjugated secondary antibody (1:5000-1:10000) for 1 hour at room temperature.

• Wash 3 times with TBST, 5 minutes each.

Detection and Analysis:

• Apply ECL substrate and detect signal using film or digital imaging system.

• Expected band size for KRT14 is approximately 50 kDa .

• Always include positive control samples known to express KRT14.

• For quantification, normalize KRT14 signal to appropriate loading control.

Troubleshooting Tips:

• If detecting endogenous KRT14, include epithelial cell line lysates as positive controls.

• For weak signals, consider extended primary antibody incubation or signal enhancement systems.

• High background may indicate need for more stringent washing or increased blocking duration.

• Multiple bands may indicate degradation products or post-translational modifications.

This protocol can be adapted based on specific sample types and equipment availability while maintaining the critical parameters for reliable KRT14 detection.

What is the optimal immunohistochemistry protocol for KRT14 detection in FFPE tissues?

The following protocol provides a standardized approach for reliable KRT14 detection in FFPE tissues:

Materials Required:

• KRT14 monoclonal antibody (recommended dilution 1:50-1:100)

• Positive control tissue: tonsil

• Negative control tissue: non-epithelial tissue

• Antigen retrieval buffer (citrate buffer pH 6.0 or EDTA buffer pH 9.0)

• Detection system: avidin-biotin complex or polymer-based system

• DAB chromogen

• Hematoxylin counterstain

Protocol Steps:

• Deparaffinization and Rehydration:

  • Heat slides at 60°C for 20 minutes

  • Xylene: 3 changes, 5 minutes each

  • 100% ethanol: 2 changes, 3 minutes each

  • 95% ethanol: 3 minutes

  • 70% ethanol: 3 minutes

  • Rinse in distilled water

• Antigen Retrieval:

  • Heat-induced epitope retrieval in pressure cooker

  • Immerse slides in preheated buffer

  • Maintain at high pressure for 3 minutes

  • Allow to cool for 20 minutes

  • Rinse in PBS, 3 changes

• Blocking and Primary Antibody:

  • Block endogenous peroxidase with 3% H₂O₂, 10 minutes

  • Rinse in PBS, 3 changes

  • Apply protein block, 10 minutes

  • Drain and apply KRT14 antibody at 1:50-1:100 dilution

  • Incubate in humidity chamber for 1 hour at room temperature or overnight at 4°C

• Detection:

  • Rinse in PBS, 3 changes

  • Apply appropriate secondary antibody, 30 minutes

  • Rinse in PBS, 3 changes

  • Apply detection reagents per manufacturer's protocol

  • Develop with DAB for 5-10 minutes (monitor microscopically)

  • Rinse in distilled water

• Counterstaining and Mounting:

  • Counterstain with hematoxylin, 30 seconds to 1 minute

  • Rinse in running tap water

  • Dehydrate through graded alcohols and xylene

  • Mount with permanent mounting medium

Expected Results:

• Strong cytoplasmic staining in basal cells of stratified epithelia

• Positive staining in myoepithelial cells in glandular tissues

• Negative staining in non-epithelial tissues

Quality Control:

• Tonsil should show strong staining in crypt epithelium

• Include antibody diluent-only negative control

• Check for non-specific background staining

This protocol can be modified for specific research needs, but any changes should be validated against the standard protocol using appropriate controls.