KRT14 Antibody

What is KRT14 and what is its biological significance?

KRT14 (Keratin 14) is a type I intermediate filament protein of approximately 51.6 kilodaltons that forms part of the cytoskeletal structure in epithelial cells. It is primarily expressed in the basal cells of stratified epithelia, including the skin, and in basal-like subtypes of certain cancers . KRT14 partners with Keratin 5 (KRT5) to form heterodimers that are essential for maintaining cellular structural integrity.

The biological significance of KRT14 includes:

• Providing mechanical support and resilience to epithelial cells

• Playing crucial roles in cell signaling and differentiation processes

• Serving as a diagnostic marker for specific epithelial cell types and cancers

• Mutations in KRT14 are associated with epidermolysis bullosa simplex, a skin fragility disorder

Which tissues and cell types normally express KRT14?

KRT14 expression follows a specific pattern in normal tissues:

Positive KRT14 Expression:

• Basal cells of stratified epithelia

• Squamous epithelia (skin, esophagus, cervix)

• Hair follicles

• Salivary glands

• Basal cells of prostate

• Basal cells of respiratory epithelium

• Amnion and chorion cells of the placenta

Negative KRT14 Expression:

• Lung parenchyma

• Liver

• Pancreas

• Epididymis

• Testis

• Urothelium

• Gastrointestinal epithelial cells

• Brunner glands

• Fallopian tube

• Trophoblastic cells of the placenta

• Adrenal and parathyroid glands

• Brain tissues

• Hematopoietic cells

• Mesenchymal tissues

This expression pattern is supported by both immunohistochemical findings and RNA expression data from the Human Protein Atlas, FANTOM5, and GTEx projects .

What are the optimal applications for KRT14 antibodies in research?

KRT14 antibodies perform reliably in the following applications:

For optimal results, validation in your specific experimental system is recommended, as antibody performance can vary between different clones and suppliers .

How do I choose between monoclonal and polyclonal KRT14 antibodies?

The choice between monoclonal and polyclonal KRT14 antibodies depends on your specific research needs:

Monoclonal Antibodies (e.g., RCK107, LL002):

• Advantages: High specificity, consistent lot-to-lot performance, reduced background

• Best for: Clinical diagnostics, quantitative studies, scenarios requiring high reproducibility

• Recommended for: IHC applications in tumor classification

Polyclonal Antibodies:

• Advantages: Recognize multiple epitopes, higher sensitivity, better for detecting denatured proteins

• Best for: Western blotting, detecting low-abundance targets, applications with potentially altered epitopes

• Recommended for: Initial protein characterization studies, WB applications

When selecting an antibody, also consider:

• Species reactivity needed (human, mouse, rat, etc.)

• Required applications (different antibodies may perform better in WB vs. IHC)

• Epitope location (N-terminal, C-terminal, or internal regions)

What are the best validation methods for KRT14 antibodies?

Proper validation ensures reliable results with KRT14 antibodies:

• Positive and negative tissue controls:

  • Use tissues with known KRT14 expression (e.g., skin) as positive controls

  • Use KRT14-negative tissues (e.g., liver, lung) as negative controls

• Knockout/knockdown validation:

  • Compare staining between wild-type cells and KRT14 knockout cells

  • KRT14 knockout cells show significantly reduced or absent staining

• Western blot confirmation:

  • Verify a single band at approximately 50 kDa (the expected molecular weight of KRT14)

• Antibody comparison:

  • Test multiple antibodies against the same samples

  • Look for consistent staining patterns across different antibody clones

• RNA correlation:

  • Compare antibody staining with RNA expression data

  • Good KRT14 antibodies show strong concordance with RNA expression patterns

How can KRT14 antibodies be used to identify "leader cells" in cancer invasion?

KRT14 has been identified as a marker for "leader cells" in ovarian cancer invasion, representing a significant application in cancer research:

Methodology for leader cell identification:

• Isolation of migratory cells:

  • Use Transwell migration assays or RTCA CIM plates to separate migratory from non-migratory cancer cells

  • Collect cells from the underside of migration membranes for analysis

• KRT14 expression analysis:

  • Compare KRT14 levels between migratory and non-migratory populations

  • Quantify using qRT-PCR, immunofluorescence, or flow cytometry

  • Research shows KRT14 mRNA is significantly enriched in migratory cells compared to pre-migratory samples

• Functional validation:

  • Generate KRT14 knockout and KRT14 overexpression cell lines

  • Assess the effect on spheroid formation and mesothelial clearance

  • KRT14 knockout cells show impaired spheroid formation initially, while KRT14 overexpression enhances dense and compact sphere formation with visible outgrowth

• Co-culture experiments:

  • Co-culture cancer spheroids with mesothelial cells

  • Measure attachment and clearance capabilities

  • Compare wild-type, KRT14 knockout, and KRT14 overexpression conditions

This approach has demonstrated that KRT14-positive cells are crucial for tumor invasion processes, suggesting their potential as therapeutic targets or prognostic markers .

What are the optimal co-immunoprecipitation protocols for studying KRT14 protein interactions?

KRT14 engages in important protein interactions, such as with RIPK4, which may regulate keratin turnover during keratinocyte differentiation . Here's an optimized co-immunoprecipitation (co-IP) protocol:

Materials needed:

• Anti-KRT14 antibody (preferably monoclonal for specificity)

• Protein A/G beads

• Cell lysis buffer (non-denaturing)

• Wash buffers

• SDS-PAGE materials

Protocol steps:

• Cell preparation:

  • Culture relevant keratinocyte cell lines (e.g., HaCaT cells)

  • For overexpression studies, transfect cells with tagged constructs (e.g., GST-KRT14, FLAG-RIPK4)

• Cell lysis:

  • Lyse cells in non-denaturing buffer containing:

    • 50 mM Tris-HCl pH 7.5

    • 150 mM NaCl

    • 1% NP-40 or 0.5% Triton X-100

    • Protease and phosphatase inhibitors

  • Perform lysis on ice for 30 minutes with gentle agitation

  • Centrifuge at 14,000 × g for 15 minutes at 4°C

• Pre-clearing (reduces non-specific binding):

  • Incubate lysate with Protein A/G beads for 1 hour at 4°C

  • Remove beads by centrifugation

• Immunoprecipitation:

  • Add 2-5 μg of anti-KRT14 antibody to pre-cleared lysate

  • Incubate overnight at 4°C with gentle rotation

  • Add fresh Protein A/G beads and incubate for 2-4 hours at 4°C

  • Collect beads by centrifugation and wash 4-5 times with wash buffer

• Elution and analysis:

  • Elute proteins by boiling in SDS sample buffer

  • Separate by SDS-PAGE and analyze by Western blotting

  • Probe with antibodies against potential interaction partners

For KRT14-RIPK4 interaction specifically, both proteins can be cloned into appropriate expression vectors (e.g., GST-KRT14 and FLAG-RIPK4) to facilitate detection of the interaction .

How can I optimize dual immunofluorescence protocols using KRT14 antibodies?

Dual immunofluorescence with KRT14 antibodies allows visualization of its relationship with other proteins. This is particularly valuable for studying epithelial architecture and cellular interactions.

Optimized protocol:

• Sample preparation:

  • Fix cells/tissues with 4% paraformaldehyde in PBS for 10 minutes

  • Permeabilize with 0.2% Triton X-100 in PBS for 5 minutes

  • Wash thoroughly (5× with PBS) between steps

• Blocking:

  • Block with 10% goat serum in PBS for 30-60 minutes at room temperature

  • This reduces non-specific binding

• Primary antibody incubation:

  • Dilute KRT14 antibody and the second target antibody in 10% goat serum

  • For KRT14: Use at 1:100-1:200 dilution

  • Ensure primary antibodies are from different host species (e.g., rabbit anti-KRT14 and mouse anti-second target)

  • Incubate for 90 minutes at room temperature or overnight at 4°C

• Secondary antibody incubation:

  • Wash 5× with PBS

  • Use species-specific secondary antibodies with distinct fluorophores

  • For example: Anti-mouse-Cy3 (1:1500) and anti-rabbit Alexa Fluor 488 (1:1000)

  • Incubate for 90 minutes at room temperature

• Nuclear counterstaining and mounting:

  • Counterstain nuclei with DAPI (1:1000) for 5 minutes

  • Mount with anti-fade mounting medium

Tips for successful dual staining:

• Validate antibodies individually before performing dual staining

• Include appropriate controls (single stains, no primary antibody controls)

• Select fluorophores with minimal spectral overlap

• When studying KRT14 with other keratins, be particularly careful about cross-reactivity

What methods are most effective for quantitative analysis of KRT14 expression in heterogeneous tissues?

Quantitative analysis of KRT14 expression in heterogeneous tissues requires specialized approaches:

1. Digital Pathology and Image Analysis:

• Scan immunostained slides using digital slide scanners

• Use image analysis software to:

  • Identify KRT14-positive cells based on staining intensity

  • Quantify percentage of positive cells

  • Measure staining intensity (low, medium, high)

  • Assess distribution patterns within the tissue

2. Tissue Microdissection and Molecular Analysis:

• Use laser capture microdissection to isolate KRT14-positive regions

• Extract RNA/protein from microdissected samples

• Quantify KRT14 expression by qRT-PCR or Western blotting

• Compare with adjacent KRT14-negative regions

3. Single-cell Analysis Approaches:

• Prepare single-cell suspensions from heterogeneous tissues

• Perform intracellular staining for KRT14

• Analyze by flow cytometry to quantify:

  • Percentage of KRT14+ cells

  • Expression level (Mean Fluorescence Intensity)

  • Co-expression with other markers

4. Multiplexed Immunofluorescence:

• Perform multiplexed staining (KRT14 plus 3-5 additional markers)

• Image using multispectral imaging systems

• Apply computational analysis to:

  • Identify cell types

  • Quantify marker expression

  • Map spatial relationships

5. Data Representation and Analysis:

How do post-translational modifications of KRT14 affect antibody binding and experimental outcomes?

Post-translational modifications (PTMs) of KRT14 can significantly impact antibody binding and experimental results:

Common PTMs affecting KRT14:

• Phosphorylation (especially during stress responses)

• Glycosylation

• Ubiquitination (during degradation)

• Acetylation

• SUMOylation

Effects on antibody binding:

• Epitope masking:

  • PTMs can physically block antibody access to epitopes

  • Phosphorylation of sites within or adjacent to the antibody epitope may prevent recognition

  • Solution: Use antibodies raised against different epitopes or specific PTM-state antibodies

• Conformational changes:

  • PTMs can alter KRT14 protein folding and structure

  • This may expose or hide epitopes recognized by certain antibodies

  • Solution: Use denaturing conditions for applications like Western blotting

• Altered migration patterns:

  • Phosphorylation typically causes slower migration on SDS-PAGE

  • May result in band shifts from the expected 50 kDa

  • Solution: Treat samples with phosphatases before SDS-PAGE if phosphorylation is suspected

Recommendations for experiments:

• Selection of appropriate antibodies:

  • For general KRT14 detection: Use antibodies targeting conserved regions less likely to be modified

  • For PTM studies: Use modification-specific antibodies (e.g., phospho-KRT14)

• Sample preparation considerations:

  • Include phosphatase inhibitors in lysis buffers to preserve phosphorylation state

  • Consider native vs. denaturing conditions based on experimental goals

• Control experiments:

  • Treat samples with enzymes that remove specific PTMs

  • Compare antibody reactivity before and after treatment

  • Include both negative and positive controls with known PTM status

Understanding the impact of PTMs on KRT14 is particularly important when studying cellular stress responses, as keratin phosphorylation often increases during stress conditions.

What are the common causes of false-positive and false-negative results with KRT14 antibodies?

Identifying and resolving false results is crucial for reliable KRT14 detection:

False-positive causes and solutions:

• Cross-reactivity with other keratins:

  • Problem: Antibodies may recognize similar epitopes in other keratins (especially type I keratins)

  • Solution: Use validated monoclonal antibodies with confirmed specificity; include negative control tissues

• Excessive antibody concentration:

  • Problem: Too high concentration leads to non-specific binding

  • Solution: Perform antibody titration to determine optimal concentration; typically 1:100-1:200 for IHC/IF

• Inadequate blocking:

  • Problem: Insufficient blocking allows non-specific binding

  • Solution: Use 10% serum from the same species as the secondary antibody; block for at least 30 minutes

• Endogenous peroxidase activity:

  • Problem: Can cause background in IHC using HRP-based detection

  • Solution: Block endogenous peroxidase with H₂O₂ treatment before antibody incubation

False-negative causes and solutions:

• Epitope masking during fixation:

  • Problem: Overfixation can cross-link proteins and hide epitopes

  • Solution: Optimize fixation time; perform antigen retrieval (heat-induced or enzymatic)

• Inappropriate antigen retrieval:

  • Problem: Insufficient retrieval of epitopes in FFPE tissues

  • Solution: Test different retrieval methods (citrate, EDTA, or Tris buffers at various pH)

• Degraded antibody:

  • Problem: Repeated freeze-thaw cycles or improper storage

  • Solution: Aliquot antibodies upon receipt; avoid repeated freeze-thaw cycles

• Sample-specific issues:

  • Problem: Heterogeneous expression or low abundance in certain samples

  • Solution: Use amplification systems (tyramide signal amplification) for low-expressing samples

Validation checklist:

• Test antibody on positive and negative control tissues

• Compare staining pattern with literature and Human Protein Atlas data

• Confirm specificity using knockout/knockdown controls when possible

• Verify expected molecular weight in Western blots (~50 kDa)

What are the optimal protocols for KRT14 detection in formalin-fixed paraffin-embedded (FFPE) tissues?

Detection of KRT14 in FFPE tissues requires optimized protocols for successful immunohistochemistry:

Standard protocol for FFPE tissues:

• Deparaffinization and rehydration:

  • Heat slides at 60°C for 1 hour

  • Deparaffinize in xylene (3 × 5 minutes)

  • Rehydrate through graded alcohols to water

• Antigen retrieval:

  • Heat-induced epitope retrieval (HIER) using:

    • Citrate buffer (pH 6.0) or EDTA buffer (pH 9.0)

    • Pressure cooker: 125°C for 30-40 seconds or

    • Microwave: 20 minutes at sub-boiling temperature

  • Cool slides to room temperature (20 minutes)

• Blocking steps:

  • Endogenous peroxidase block: 3% H₂O₂ for 10 minutes

  • Protein block: 10% normal goat serum for 30 minutes

• Primary antibody incubation:

  • Dilute KRT14 antibody 1:100-1:200 in antibody diluent

  • Incubate overnight at 4°C or 60 minutes at room temperature

• Detection system:

  • Polymer-based detection systems generally work well

  • For chromogenic detection: DAB substrate (brown)

  • For fluorescent detection: Use fluorophore-conjugated secondary antibodies

  • Counterstain with hematoxylin (for chromogenic) or DAPI (for fluorescent)

Key optimization considerations:

• Antigen retrieval method: Test both citrate and EDTA-based buffers to determine optimal retrieval

• Antibody dilution: Titrate antibody to find optimal concentration for your specific samples

• Incubation time and temperature: Adjust based on staining intensity and background

• Detection system sensitivity: Consider amplification systems for weak expression

Expected results:

• KRT14-positive cells show cytoplasmic/membrane staining

• Positive control (skin) should show strong basal cell staining

• Negative controls (liver, lung) should show no specific staining

How can I optimize KRT14 antibody protocols for flow cytometry applications?

Flow cytometry with KRT14 antibodies requires special considerations due to its intracellular location:

Optimized protocol for flow cytometry:

• Cell preparation:

  • Harvest cells (enzymatic or mechanical dissociation)

  • Wash in cold PBS with 2% FBS

  • Adjust to 1 × 10⁶ cells per sample

• Fixation and permeabilization:

  • Fix cells with 4% paraformaldehyde for 15 minutes at room temperature

  • Wash twice with PBS

  • Permeabilize with 0.1% Triton X-100 or commercial permeabilization buffer for 15 minutes

  • Critical step: Adequate permeabilization is essential for antibody access to intracellular KRT14

• Blocking:

  • Block with 10% serum (from secondary antibody species) for 30 minutes

  • This reduces non-specific binding

• Antibody staining:

  • Primary antibody: Anti-KRT14 at 1:100-1:200 dilution for 45-60 minutes

  • Wash three times with PBS containing 2% FBS

  • If using unconjugated primary antibody:

    • Add fluorophore-conjugated secondary antibody (1:500)

    • Incubate 30 minutes in the dark

    • Wash three times

• Analysis considerations:

  • Use appropriate compensation controls if multiplexing

  • Include FMO (Fluorescence Minus One) controls

  • Use isotype controls to assess background staining

Troubleshooting flow cytometry issues:

Dual staining strategies:
For co-expression analysis, consider these marker combinations:

• KRT14 + KRT5 (basal cell identification)

• KRT14 + Proliferation markers (Ki67)

• KRT14 + Differentiation markers (Involucrin)

• KRT14 + Cancer stem cell markers (CD44, ALDH)

What strategies can improve KRT14 antibody performance in challenging sample types?

Certain sample types present challenges for KRT14 detection, requiring specialized approaches:

1. Highly keratinized tissues (e.g., skin):

• Challenge: High background due to endogenous keratins

• Solutions:

  • Extended blocking (2-3 hours) with 10% serum + 1% BSA

  • Use monoclonal antibodies with high specificity

  • Include highly stringent washing steps (0.1% Tween-20 in PBS)

  • Pre-absorb antibodies with keratin-rich extracts

2. Archival FFPE tissues:

• Challenge: Epitope degradation or masking

• Solutions:

  • Extended antigen retrieval (30-40 minutes)

  • Test multiple antibody clones targeting different epitopes

  • Use signal amplification systems (tyramide signal amplification)

  • Consider dual antibody approach (cocktail of two KRT14 antibodies)

3. Frozen tissue sections:

• Challenge: Compromised morphology, high background

• Solutions:

  • Optimal cutting temperature (8-10 μm sections)

  • Post-fixation with acetone (10 minutes at -20°C)

  • Pre-treatment with 0.3% Triton X-100 (5 minutes)

  • Extended washing steps to remove OCT compound

4. Cell cultures with low KRT14 expression:

• Challenge: Weak signal, difficult detection

• Solutions:

  • Use high-sensitivity detection systems

  • Longer primary antibody incubation (overnight at 4°C)

  • Consider more sensitive antibody clones

  • Use signal amplification techniques

5. Tissue microarrays (TMAs):

• Challenge: Heterogeneous expression, small sample size

• Solutions:

  • Optimize protocols on whole sections before TMA analysis

  • Include positive control cores

  • Consider duplicate/triplicate cores for each sample

  • Adjust scoring systems to account for heterogeneity

For all challenging samples, pilot studies with multiple antibody clones, dilutions, and detection systems are recommended to determine optimal conditions.

How can KRT14 antibodies be used effectively in cancer research?

KRT14 antibodies have diverse applications in cancer research, particularly for studying tumor progression and classification:

1. Tumor classification and subtyping:

• Methodology: IHC staining of tissue microarrays or whole sections

• Applications:

  • Identification of basal-like breast cancers (KRT14+/KRT5/6+)

  • Characterization of squamous cell carcinomas

  • Classification of urothelial carcinomas

  • Distinguishing subtypes with different prognoses

2. Identification of cancer stem cells and leader cells:

• Methodology: Dual immunofluorescence, flow cytometry, or single-cell sequencing

• Applications:

  • Isolation of KRT14+ cells from heterogeneous tumors

  • Functional characterization of their invasion and migration abilities

  • KRT14+ cells in ovarian cancer function as "leader cells" that facilitate invasion

3. Epithelial-mesenchymal transition (EMT) studies:

• Methodology: Sequential staining of tumor sections or time-course analysis

• Applications:

  • Track changes in KRT14 expression during EMT

  • Correlate with expression of EMT markers (E-cadherin, Vimentin)

  • Study partial EMT states in collective invasion

4. Therapeutic response prediction:

• Methodology: Pre- and post-treatment biopsy analysis

• Applications:

  • Assess changes in KRT14+ cell populations after therapy

  • Correlate KRT14 expression with resistance to specific treatments

  • Identify KRT14+ residual disease after treatment

5. Metastasis research:

• Methodology: Comparative analysis of primary tumors and metastatic sites

• Applications:

  • Determine if KRT14+ cells are enriched in metastatic lesions

  • Study if KRT14 expression changes during metastatic colonization

  • Evaluate the role of KRT14+ cells in metastasis initiation

Research protocol example: Analyzing KRT14+ leader cells in invasion assays

• 3D spheroid invasion assay:

  • Generate spheroids from cancer cells in low-adhesion plates

  • KRT14 knockout, wild-type, and KRT14 overexpression cells show different spheroid formation kinetics

  • Embed spheroids in collagen matrix

  • Track invasion over 24-72 hours

  • Fix and stain for KRT14 to identify leading cells

• Mesothelial clearance assay:

  • Culture mesothelial cell monolayer to confluence

  • Add tumor spheroids to the monolayer

  • Measure clearance area over time

  • Compare clearance ability between KRT14 knockout and wild-type cells

  • Reveals functional importance of KRT14 in invasion processes

What are the best methods for studying KRT14 interactions with other proteins?

Understanding KRT14's interactions with other proteins provides insights into its functions beyond structural support:

1. Proximity Ligation Assay (PLA):

• Principle: Detects protein interactions in situ with single-molecule sensitivity

• Methodology:

  • Incubate samples with primary antibodies against KRT14 and partner protein

  • Add PLA probes (secondary antibodies with oligonucleotide tags)

  • If proteins are in proximity (<40 nm), oligonucleotides can be ligated

  • Amplify signal by rolling circle amplification

  • Detect fluorescent spots indicating interaction sites

2. FRET (Förster Resonance Energy Transfer):

• Principle: Energy transfer between fluorophores when proteins are in close proximity

• Methodology:

  • Label KRT14 and interaction partner with donor/acceptor fluorophores

  • Measure energy transfer using specialized microscopy

  • Calculate FRET efficiency to quantify interaction strength

  • Perform acceptor photobleaching to confirm specificity

3. Co-Immunoprecipitation with specific controls:

• Principle: Physical isolation of protein complexes

• Methodology:

  • Use stringent lysis conditions that preserve keratin interactions

  • Include cytoskeletal stabilizing buffers

  • Perform reciprocal IPs (pull down with anti-KRT14 and partner antibody)

  • Include controls for non-specific binding

  • Analyze by mass spectrometry for unbiased interaction discovery

4. Yeast Two-Hybrid (Y2H) screening:

• Principle: Identifies direct protein-protein interactions

• Methodology:

  • Clone KRT14 as bait (e.g., in fusion with Gal4 DNA binding domain)

  • Screen against human keratinocyte cDNA library

  • Identify positive interactions through reporter activation

  • Verify interactions using different Y2H systems

  • This approach has successfully identified KRT14-RIPK4 interactions

5. Bimolecular Fluorescence Complementation (BiFC):

• Principle: Protein interaction brings together fragments of fluorescent protein

• Methodology:

  • Fuse KRT14 and partner protein with complementary fragments of fluorescent protein

  • Co-express in relevant cell types

  • Interaction brings fragments together, reconstituting fluorescence

  • Visualize by fluorescence microscopy

Example: KRT14-RIPK4 interaction study methodology

• Clone KRT14 into GST-tagged expression vectors and RIPK4 into FLAG-tagged vectors

• Express in HeLa or HaCaT cells

• Perform co-immunoprecipitation with anti-FLAG antibodies

• Detect pulled-down KRT14 using anti-GST antibodies

• Confirm by reverse IP and fluorescence co-localization

• This approach revealed that RIPK4 may regulate keratin turnover required for keratinocyte differentiation

How can KRT14 antibodies be integrated into single-cell analysis workflows?

Integrating KRT14 antibodies into single-cell technologies enables unprecedented insights into cellular heterogeneity:

1. Single-cell proteomics with antibody-based approaches:

• CITE-seq (Cellular Indexing of Transcriptomes and Epitopes by Sequencing):

  • Label cells with oligo-tagged KRT14 antibodies

  • Perform single-cell RNA sequencing

  • Simultaneously measure KRT14 protein and transcriptome

  • Reveals potential post-transcriptional regulation

• CyTOF (Mass cytometry):

  • Label with metal-conjugated KRT14 antibodies

  • Allows simultaneous detection of 40+ proteins

  • No fluorescence overlap issues

  • Ideal for deep phenotyping of KRT14+ subpopulations

2. Spatial transcriptomics with protein detection:

• Visium with immunofluorescence:

  • Perform KRT14 immunostaining on tissue sections

  • Capture spatial transcriptomics data

  • Correlate KRT14 protein expression with local transcriptome

  • Reveals tissue microenvironments around KRT14+ cells

• CODEX (CO-Detection by indEXing):

  • Multiplex KRT14 with 40+ other antibodies

  • Preserves spatial context

  • Reveals complex cellular neighborhoods

  • Ideal for tumor microenvironment studies

3. Live-cell imaging approaches:

• Antibody fragments for live imaging:

  • Use fluorescently labeled Fab fragments of KRT14 antibodies

  • Track dynamics in living cells

  • Minimal interference with protein function

  • Enables study of KRT14 dynamics during migration

4. In situ sequencing with protein detection:

• MERFISH with antibody detection:

  • Combine in situ RNA detection with KRT14 antibody staining

  • Visualize both transcripts and protein in the same cells

  • Assess correlation between mRNA and protein levels

  • Reveals post-transcriptional regulation mechanisms

Practical considerations for single-cell applications:

• Antibody validation is critical for single-cell applications

• Titrate antibodies to minimize background

• Include appropriate controls (isotype, FMO)

• Consider clone-specific performance in different applications

• Validate staining on known positive and negative populations

What are the critical controls needed when studying KRT14 knockout or knockdown models?

1. Validation of knockout/knockdown efficiency:

• Protein level validation:

  • Western blotting with at least two different KRT14 antibodies targeting different epitopes

  • Immunofluorescence to assess cellular distribution and expression level

  • Flow cytometry for quantitative assessment of population-level changes

• Transcript level validation:

  • qRT-PCR with primers spanning different exons

  • RNA-seq to confirm complete absence of KRT14 transcripts

  • Assessment of potential compensatory changes in other keratins

2. Essential experimental controls:

• Genetic controls:

  • Non-targeting CRISPR control (for CRISPR-Cas9 knockout)

  • Scrambled siRNA/shRNA (for knockdown approaches)

  • Empty vector control (for overexpression studies)

  • Rescue experiments (re-expression of KRT14 in knockout cells)

• Technical controls:

  • Multiple independent knockout/knockdown clones to avoid clone-specific effects

  • Mixed population analysis to assess heterogeneous responses

  • Time-course analysis to distinguish acute vs. adaptative responses

3. Phenotypic assessment controls:

• Functional assays:

  • Spheroid formation assays show different kinetics between KRT14 knockout and wild-type cells

  • Migration/invasion assays with appropriate ECM components

  • Comparison of different assay types (2D vs. 3D) to validate findings

• Molecular profiling:

  • Assessment of other keratin expression (potential compensation)

  • Analysis of keratin-binding proteins

  • Evaluation of cell-type-specific markers to confirm cell identity

4. Specialized controls for specific applications:

• For in vivo studies:

  • Conditional knockout models to avoid developmental effects

  • Tissue-specific promoters to target specific cell populations

  • Littermate controls with matched genetic background

  • Assessment of immune cell infiltration as potential confounder

• For therapeutic targeting:

  • Dose-response studies to establish specificity

  • Off-target effect assessment

  • Rescue experiments with modified KRT14 constructs

  • Comparison with other cytoskeletal disruption approaches

Example from research: In a study of KRT14's role in ovarian cancer invasion, researchers generated both KRT14 knockout and overexpression cell lines, comparing them to wild-type and non-targeting CRISPR control cells. The phenotypic differences were assessed through spheroid formation assays, which revealed that KRT14 knockout cells showed impaired spheroid formation initially but formed comparable spheroids after extended incubation. Conversely, KRT14 overexpression lines formed dense spheroids rapidly with visible outgrowth .