KRT14 Antibody

What is KRT14 and why is it important as a research target?

KRT14 (Keratin 14) is a 51.6 kDa protein belonging to the type I (acidic) subfamily of low molecular weight keratins that exists in combination with keratin 5 (type II/basic) . It is predominantly expressed in the basal cells of stratified epithelia, including the epidermis, as well as in some glandular epithelia, myoepithelium, and mesothelial cells .

KRT14's importance as a research target stems from its critical functions:

• Providing mechanical strength to epithelial cells and protecting them from mechanical stress

• Playing key roles in cell signaling, adhesion, and migration

• Serving as a diagnostic marker for epithelial tumors and tissue regeneration

• Involvement in disease pathogenesis, as mutations in the KRT14 gene are associated with epidermolysis bullosa simplex (EBS), a condition characterized by skin blistering and erosion

KRT14 expression is also dynamically regulated during tissue regeneration and in various disease states, making it valuable for studying these processes.

What are the key considerations when selecting a KRT14 antibody?

When selecting a KRT14 antibody for research, several factors should be considered:

Antibody Type:

• Monoclonal antibodies (e.g., clones LL002, RCK107, KRT14-2375) offer high specificity for a single epitope but may be sensitive to epitope masking

• Polyclonal antibodies recognize multiple epitopes, potentially providing stronger signals but with potential for higher background

Target Region:

• Some antibodies target the C-terminus of KRT14

• Others target specific amino acid sequences (e.g., AA 351-472, AA 276-303)

• The target region may affect accessibility in fixed tissues or compatibility with specific applications

Species Reactivity:

• Consider cross-reactivity with orthologs from relevant experimental models (human, mouse, rat, pig, dog)

• Canine, porcine, monkey, mouse, and rat orthologs may be detected by some antibodies

Application Compatibility:

• Verify validation for your specific application (WB, IHC, IF, FACS, ICC)

• Some antibodies work better for particular applications (e.g., paraffin sections vs. frozen sections)

Clone Selection:
The table below summarizes properties of common KRT14 antibody clones:

What protocols yield optimal results for KRT14 immunostaining?

Effective KRT14 immunostaining requires attention to several methodological factors:

Sample Preparation:

• For tissues: Standard fixation with 10% neutral buffered formalin works well for most KRT14 antibodies

• For cells: 4% paraformaldehyde for 15-20 minutes at room temperature preserves KRT14 architecture

Antigen Retrieval:

• Heat-induced epitope retrieval using citrate buffer (pH 6.0) or EDTA buffer (pH 9.0)

• For some antibodies, enzymatic retrieval may be necessary (consult specific antibody documentation)

Recommended Dilutions:

• For IHC: Typically 1:20-1:200 depending on the specific antibody

• Always perform an antibody titration to determine optimal concentration

Controls:

• Positive controls: HeLa, A549 or A431 cells; skin or squamous cell carcinoma

• Negative controls: Tissues known not to express KRT14; isotype control antibodies

Detection Systems:

• For bright-field microscopy: HRP-based detection systems work well

• For fluorescence: Consider secondary antibodies with bright fluorophores like Alexa Fluor dyes

Special Considerations:

• For spheroids or 3D cultures: Extend incubation times and use higher antibody concentrations to ensure penetration

• For dual staining with other keratins: Use sequentially rather than simultaneously to avoid potential cross-reactivity

How can KRT14 antibodies be validated to ensure specificity?

Thorough validation of KRT14 antibodies is crucial to ensure experimental rigor:

Western Blotting:

• Should detect a single band at approximately 51.6 kDa in positive control lysates

• Consider using recombinant KRT14 protein as a positive control

Genetic Models:

• Use KRT14 knockout cells or tissues as negative controls

• Research shows that CRISPR-based Krt14-KO can effectively eliminate KRT14 expression

Peptide Competition:

• Pre-incubating the antibody with excess KRT14 peptide should abolish specific staining

Multiple Antibody Approach:

• Use antibodies from different clones targeting different epitopes

• Concordant results increase confidence in specificity

Orthogonal Methods:

• Correlate protein detection with mRNA expression data

• Consider in situ hybridization to confirm expression patterns

Tissue Distribution Analysis:

• Verify that staining matches the known tissue distribution (basal cells of stratified epithelia)

• Absence of staining in tissues known not to express KRT14

What are the common artifacts and pitfalls when using KRT14 antibodies?

Researchers should be aware of several potential issues:

Non-specific Binding:

• Some antibodies may cross-react with other keratins, particularly other type I keratins

• Careful blocking (3-5% BSA or normal serum) can minimize this issue

Epitope Masking:

• Fixation can mask KRT14 epitopes, making them inaccessible to antibodies

• Proper antigen retrieval and optimization of fixation protocols are essential

Edge Effects in Tissue Sections:

• Enhanced staining at tissue edges due to better antibody penetration

• Can be minimized by ensuring even fixation and proper tissue processing

Autofluorescence:

• Especially problematic in tissues like skin with high collagen content

• Use specific quenching methods or spectral unmixing to differentiate signal from autofluorescence

Inconsistent Results Between Applications:

• An antibody that works well for IHC may not work for Western blotting

• Validate each antibody specifically for your intended application

Batch-to-Batch Variability:

• Especially with polyclonal antibodies

• Record lot numbers and test new lots against previous ones

How can KRT14 antibodies be used to identify and isolate specific cell populations?

KRT14 antibodies have become powerful tools for identifying and isolating specific cell populations, particularly in heterogeneous tissues:

Flow Cytometry and Cell Sorting:

• KRT14 antibodies can be used to identify and isolate basal epithelial cells

• For intracellular staining, cells must be fixed and permeabilized

• Consider using commercially available fixation/permeabilization kits optimized for cytoskeletal proteins

Lineage Tracing:

• KRT14-CreERT2 recombinase lines allow for tamoxifen-inducible genetic labeling of KRT14+ cells

• Research shows these cells can be traced through development, regeneration, and disease processes

Stem Cell Identification:

• In many epithelia, KRT14+ cells represent stem/progenitor populations

• Studies show KRT14+ cells have higher proliferative potential during tissue regeneration

• The mitotic index of KRT14+ cells is approximately threefold higher than KRT14- cells during early regeneration

Cancer Stem Cell Research:

• KRT14+ cells may represent cancer stem cells in certain tumors

• In ovarian cancer spheroids, KRT14+ cells are exclusively localized to the outer spheroid rim

Isolation Protocol Considerations:

• For live cell isolation, reporter systems (e.g., GFP knocked into the KRT14 locus) are preferable

• When using antibody-based isolation, gentle enzymatic dissociation methods preserve KRT14 epitopes

• Magnetic bead separation can be less damaging than FACS for sensitive epithelial cells

What are the dynamics of KRT14 expression during tissue regeneration and wound healing?

KRT14 expression is dynamically regulated during tissue repair and regeneration, making it a valuable marker for studying these processes:

Baseline Expression:

• Under normal conditions, KRT14 expression is restricted to basal cells in stratified epithelia

• In many tissues, only a small percentage of cells are KRT14+

Response to Injury:

• Following tissue damage, a marked increase in KRT14+ cell numbers occurs

• In bladder regeneration models, KRT14+ cells increase to 22.3±2.2% of cells at 48h post-injury

• This increase coincides with the active phase of tissue regeneration

Proliferative Dynamics:

• KRT14+ cells show higher proliferative activity compared to KRT14- cells during regeneration

• Between 18-24h post-injury, the mitotic index of KRT14+ cells is approximately 3x higher than KRT14- cells

• This difference decreases to 1.4x by 48h but remains statistically significant

Fate Mapping:

• Lineage tracing experiments using KRT14-CreERT2 systems reveal the fate of KRT14+ cells

• These cells can contribute to multiple differentiated cell types during regeneration

Temporal Regulation:

• KRT14 expression peaks during active regeneration and declines after repair completion

• This suggests a feedback mechanism regulating KRT14 expression based on tissue homeostasis

How do KRT14 antibodies perform in cancer diagnostics and research?

KRT14 antibodies have significant applications in cancer research and diagnostics:

Diagnostic Applications:

• Differentiating squamous cell carcinomas from poorly differentiated epithelial tumors

• Distinguishing between basal and non-basal subtypes of breast carcinomas

• Separating oncocytic tumors of the kidney from renal mimics

• Identifying metaplastic carcinomas of the breast

Cancer Stem Cell Identification:

• In certain cancers, KRT14+ cells may represent cancer stem cell populations

• After 6 months of BBN exposure in bladder cancer models, tumors develop that almost exclusively express both Krt5 and Krt14

Tumor Invasion Mechanisms:

• KRT14+ leader cells mediate mesothelial clearance in ovarian cancer metastasis

• KRT14 is restricted to cells at the invasive edge in 3D spheroid models

Triple-Negative Breast Cancer (TNBC):

• H3K27me3-mediated KRT14 upregulation promotes TNBC metastasis

• Loss of KRT14 severely compromises TNBC peritoneal metastasis

• EZH2 (which affects H3K27me3) functional loss reduces cancer migration, invasion, and metastasis

Staining Patterns:

• Cytoplasmic staining in epithelial cells

• In invasive cancers, may show aberrant expression patterns

• Important to evaluate in conjunction with other markers for accurate diagnosis

What controls should be included when working with KRT14 antibodies?

Proper controls are essential for reliable results with KRT14 antibodies:

Positive Tissue Controls:

• Skin: Strong positivity in basal keratinocytes

• Squamous cell carcinoma: Typically shows robust KRT14 expression

• Myoepithelial cells of breast ducts: Consistent KRT14 expression

Positive Cell Line Controls:

• HeLa, A549, or A431 cells have been validated for KRT14 expression

• Each antibody datasheet should specify optimal positive controls

Negative Controls:

• Primary antibody omission: Replace primary antibody with antibody diluent

• Isotype control: Use matched isotype antibody at the same concentration

• Tissues known to lack KRT14 expression (e.g., cardiac muscle)

Genetic Controls:

• KRT14 knockout or knockdown samples when available

• Inducible systems where KRT14 expression can be modulated

Application-Specific Controls:

• For Western blots: Recombinant KRT14 protein or lysate with known KRT14 expression

• For IHC/IF: Sequential sections with and without primary antibody

• For FACS: Fluorescence minus one (FMO) controls

Validation Controls:

• Peptide competition/blocking: Pre-incubate antibody with immunizing peptide

• Multiple antibody approach: Use antibodies targeting different KRT14 epitopes

What is the relationship between KRT14 and other keratin proteins?

Understanding the relationship between KRT14 and other keratins is crucial for proper experimental design and interpretation:

KRT14 and KRT5 Partnership:

• KRT14 (type I) and KRT5 (type II) form obligate heterodimers in basal epithelial cells

• They typically co-express in the same cells, particularly in basal cells of stratified epithelia

• In bladder cancer models, Krt5 expression absolutely coincided with Krt14 expression

KRT14 and KRT15 Functional Differences:

• Despite structural similarities, KRT14 and KRT15 have distinct functions

• Krt14-KO and Krt15-KO produce contrasting phenotypes in airway basal cells :

  • Krt14-KO cells cannot differentiate into club and ciliated cells but show enhanced clonogenicity

  • Krt15-KO does not alter differentiation but impairs clonogenicity in vitro and reduces label-retaining basal cells after injury

Molecular Interactions:

• KRT14, but not KRT15, binds the tumor suppressor stratifin (Sfn)

• Disruption of KRT14 reduces Sfn protein abundance and increases expression of the oncogene dNp63a during differentiation

• KRT15-KO reduces dNp63a expression, contrasting with KRT14-KO effects

Expression Patterns:

• Under homeostatic conditions, KRT14 is expressed in basal cells of stratified epithelia

• During wound healing or cancer progression, keratin expression patterns may shift

• In some contexts, KRT14 expression can be induced in cells that normally express other keratins

Experimental Considerations:

• When studying one keratin, consider potential compensatory changes in other keratins

• Antibody cross-reactivity between keratins is a potential issue requiring careful validation

What are the challenges and strategies in targeting KRT14 for gene therapy approaches?

Targeting KRT14 for gene therapy, particularly for epidermolysis bullosa simplex (EBS), presents unique challenges and opportunities:

Challenges in KRT14 Targeting:

• Dominant-negative mutations: Many KRT14 mutations cause disease through dominant-negative effects, requiring silencing of the mutant allele

• Stem cell targeting: Long-term correction requires modification of epidermal stem cells

• Delivery efficiency: Efficient delivery to basal keratinocytes is needed for clinical benefit

• Integration specificity: Precise genomic integration is crucial to avoid disrupting other genes

Vector Systems:

• Adeno-associated virus (AAV) vectors have shown promise for KRT14 targeting

• Research demonstrates that AAV vectors can recombine with chromosomal sequences to correct mutations or disrupt production of proteins with dominant-negative activity

Gene-Targeting Strategies:

• Promoter trap design has been effective for targeting one allele of KRT14

• Targeted alteration of KRT14 resulted in functional correction in keratinocytes

• CRISPR/Cas9 approaches offer newer options for precise KRT14 editing

Stem Cell Selection:

• Successfully targeted keratinocyte stem cells with holoclone phenotype can be recovered

• These modified stem cells maintain long-term correction potential

Clinical Translation:

• Modified keratinocytes can be expanded to generate clinically significant skin grafts

• Skin equivalents from targeted cells functioned normally after transplantation, providing barrier function

• Issues of safety, efficacy, and long-term stability remain to be fully addressed

How can single-cell techniques be optimized for KRT14 detection and analysis?

Single-cell analysis of KRT14 requires specialized techniques and considerations:

Single-Cell RNA Sequencing:

• KRT14 transcripts can be detected in scRNA-seq data

• Consideration of dropout effects is important as intermediate-abundance transcripts like KRT14 may be inconsistently detected

• Specialized protocols for epithelial cells may improve detection

Single-Cell Protein Analysis:

• Antibody-based techniques like CyTOF or CODEX can quantify KRT14 at single-cell resolution

• Careful optimization of fixation and permeabilization is crucial

• Multiplexing with other epithelial markers improves cell type identification

Spatial Transcriptomics:

• Techniques like Visium or MERFISH can detect KRT14 mRNA with spatial context

• Allows correlation of KRT14 expression with microenvironmental features

Single-Cell Western Blotting:

• Can quantify KRT14 protein in individual cells

• Requires optimization of lysis conditions to solubilize intermediate filament proteins

Live Cell Imaging:

• Fluorescent protein reporters knocked into the KRT14 locus enable real-time tracking

• GFP-KRT14 fusion proteins should be validated to ensure normal function and localization

Computational Analysis:

• When analyzing KRT14+ cells, consider trajectory inference methods to map developmental paths

• Pseudotime analysis can reveal dynamics of KRT14 expression during differentiation

What is the role of KRT14-positive leader cells in cancer invasion and metastasis?

Recent research has revealed specialized functions of KRT14+ cells in cancer progression:

Leader Cell Identification:

• In various cancers, KRT14+ cells are found at the invasive front

• In ovarian cancer spheroids, KRT14 is exclusively localized to the outer spheroid rim

• These cells appear to lead collective invasion processes

Functional Significance:

• KRT14 is a critical regulator of triple-negative breast cancer (TNBC) splenic metastasis

• Loss of KRT14 severely compromises TNBC peritoneal metastasis, even in H3K27me3 hyper-activated backgrounds

• This suggests KRT14 is not merely a marker but functionally important for metastasis

Molecular Mechanisms:

• H3K27me3 mediates KRT14 upregulation by altering the recruitment pattern of its transcription factor Sp1

• EZH2, which affects H3K27me3, influences cancer migration and invasion through this pathway

• In sequential in-vivo imaging experiments, KRT14+ cells were shown to drive metastatic processes

Therapeutic Implications:

• Targeting KRT14+ leader cells could potentially inhibit metastatic spread

• The EZH2/H3K27me3/KRT14 axis may represent a therapeutic target

• EPZ6438, an H3K27me3 selective inhibitor, has shown efficacy in reducing TNBC migration, invasion, and peritoneal metastasis

Detection Methods:

• Multiplex immunofluorescence can identify KRT14+ leader cells at invasion fronts

• Single-cell RNA sequencing can characterize the transcriptional state of these cells

• Lineage tracing allows tracking of these cells during metastatic spread

How does epigenetic regulation influence KRT14 expression in development and disease?

Epigenetic mechanisms play crucial roles in regulating KRT14 expression:

H3K27me3-Mediated Regulation:

• H3K27me3 (trimethylation of lysine 27 on histone H3) influences KRT14 expression

• This histone modification alters the recruitment pattern of Sp1, a transcription factor for KRT14

• In triple-negative breast cancer, this epigenetic mechanism promotes KRT14 upregulation

EZH2 Involvement:

• EZH2 (Enhancer of Zeste Homolog 2) is a histone methyltransferase that catalyzes H3K27 trimethylation

• EZH2 functional loss (through knockdown or inhibition) significantly reduces KRT14-dependent processes

• Treatment with EPZ6438 (an H3K27me3 selective inhibitor) reduces cancer metastasis

Developmental Regulation:

• During normal development, epigenetic mechanisms ensure tissue-specific KRT14 expression

• Repressive marks like H3K27me3 prevent KRT14 expression in inappropriate cell types

• Dynamic changes in these marks accompany differentiation of epithelial progenitors

Disease-Associated Dysregulation:

• In cancer, aberrant epigenetic regulation leads to inappropriate KRT14 expression

• Epigenetic changes may precede genetic alterations in disease progression

• Understanding these mechanisms offers potential therapeutic targets

Experimental Approaches:

• ChIP-seq for histone modifications can map the epigenetic landscape at the KRT14 locus

• ATAC-seq reveals chromatin accessibility changes affecting KRT14 expression

• DNA methylation analysis can identify additional regulatory mechanisms

What are the functional differences between KRT14 and KRT15 in regulating stem cell properties?

Recent research has revealed distinct and sometimes opposing functions of KRT14 and KRT15:

Differentiation Capacity:

• Krt14-KO cells cannot differentiate into club and ciliated cells

• Krt15-KO does not alter differentiation potential

• This suggests KRT14 is required for proper differentiation while KRT15 is dispensable

Clonogenic Potential:

• Krt14-KO enhances clonogenicity (colony-forming ability)

• Krt15-KO impairs clonogenicity in vitro

• Krt15-KO reduces the number of label-retaining basal cells in vivo after injury

• These contrasting effects suggest opposing roles in regulating stem cell properties

Molecular Interactions:

• KRT14, but not KRT15, binds the tumor suppressor stratifin (Sfn)

• Disruption of KRT14 reduces Sfn protein abundance

• KRT14 disruption increases expression of the oncogene dNp63a during differentiation

• KRT15-KO reduces dNp63a levels, creating an opposing effect

Disease Relevance:

• The phenotype of Krt15-KO cells resembles the phenotype in bronchiolitis obliterans (BO)

• Both show decreased clonogenicity, increased KRT14, and decreased dNp63a expression

• This suggests KRT15 loss may contribute to pathological states where regenerative capacity is impaired

Experimental Applications:

• These differential functions can be exploited to manipulate stem cell behavior

• Transient suppression of KRT14 might enhance stem cell expansion

• Preserving KRT15 expression could help maintain regenerative capacity in transplanted cells