KRT7 Monoclonal Antibody

What is KRT7 and what is its biological significance?

KRT7 (Keratin-7) is a cytoskeletal protein primarily expressed in epithelial cells, including those lining the bladder, gastrointestinal tract, and bile ducts. Its primary function is providing structural support to these cells and maintaining their mechanical integrity. Beyond structural roles, KRT7 has demonstrated importance in cell signaling pathways, cellular differentiation processes, and migration. The protein has gained significant research interest due to its implication in various cancer types and potential role as a diagnostic marker . KRT7 has a calculated molecular weight of approximately 51 kDa, though it is commonly observed at around 40 kDa in Western blot applications .

How are KRT7 monoclonal antibodies produced for research applications?

KRT7 monoclonal antibodies are produced through hybridoma technology, a sophisticated bioengineering process. Initially, mice are immunized with synthesized peptides derived from human KRT7. B cells are then harvested from the immunized mouse spleen and fused with myeloma cells to create hybridoma cells. These hybridomas are screened to identify clones specifically producing KRT7 antibodies. Selected hybridoma cells are cultured in the mouse abdominal cavity to produce monoclonal antibodies. The final step involves purifying these antibodies from mouse ascites using affinity chromatography with the specific immunogen, ensuring high antibody purity and specificity . This rigorous production process guarantees consistency and reliability in research applications.

What are the standard applications for KRT7 monoclonal antibodies in research?

KRT7 monoclonal antibodies are validated for multiple research applications with varying recommended dilutions:

When selecting an antibody for a specific application, researchers should consider the host species (commonly rabbit or mouse), clonality (monoclonal for consistency), and validated reactivity against human, mouse, or rat KRT7 .

How should researchers optimize KRT7 antibody dilutions for immunohistochemistry applications?

Optimizing KRT7 antibody dilutions for immunohistochemistry requires a systematic approach. Begin with the manufacturer's recommended range (typically 1:20-1:200) and perform a dilution series experiment using positive control tissues known to express KRT7 (epithelial tissues from bladder, gastrointestinal tract, or bile ducts). For each dilution, evaluate signal-to-noise ratio, background staining, and specific cellular localization patterns. Include negative controls (tissues known not to express KRT7 or primary antibody omission) to assess non-specific binding. When evaluating results, look for clear cytoplasmic staining in epithelial cells with minimal background. For quantitative studies, select the dilution that provides consistent staining across replicates while maintaining specificity. Document optimization parameters including antigen retrieval methods, incubation times, and detection systems to ensure reproducibility across experiments.

What controls are essential when designing experiments using KRT7 monoclonal antibodies?

Robust experimental design with KRT7 monoclonal antibodies requires several crucial controls:

• Positive tissue controls: Include tissues with known KRT7 expression (epithelial cells from bladder, gastrointestinal tract, bile ducts) .

• Negative tissue controls: Incorporate tissues lacking KRT7 expression to verify antibody specificity.

• Technical controls:

  • Primary antibody omission control (to assess secondary antibody specificity)

  • Isotype control (matched immunoglobulin at the same concentration)

  • Blocking peptide control (pre-incubation with the immunizing peptide)

• Cellular expression controls:

  • Cell lines with confirmed KRT7 expression (positive)

  • Cell lines with confirmed KRT7 absence (negative)

  • KRT7 knockdown/knockout controls using siRNA or CRISPR technologies

These controls help distinguish between true positive signals and experimental artifacts, enhancing data reliability and facilitating accurate interpretation of experimental results.

How can researchers effectively troubleshoot weak or non-specific staining with KRT7 antibodies?

When encountering weak or non-specific staining with KRT7 antibodies, researchers should implement a structured troubleshooting approach:

For weak staining:

• Verify antibody concentration – try using a more concentrated antibody dilution within the recommended range (1:20-1:200) .

• Optimize antigen retrieval methods – heat-induced epitope retrieval with citrate buffer (pH 6.0) or EDTA buffer (pH 9.0) may improve antigen accessibility.

• Increase incubation time or temperature – extending primary antibody incubation to overnight at 4°C may enhance sensitivity.

• Evaluate detection system sensitivity – consider amplification systems like tyramide signal amplification.

• Check sample quality and fixation – overfixation can mask epitopes.

For non-specific staining:

• Increase blocking time and concentration – use 5-10% serum from the same species as the secondary antibody.

• Reduce antibody concentration – dilute primary antibody further.

• Include additional washing steps – more frequent and longer washes with PBS-T.

• Test antibody specificity – use Western blot to confirm the antibody recognizes a protein of the expected molecular weight (approximately 40 kDa) .

• Evaluate cross-reactivity – confirm antibody specificity using KRT7-null tissues or cells.

Document all optimization parameters to maintain experimental reproducibility.

How can KRT7 expression analysis be integrated with RNA-seq data to understand epithelial cancer biology?

Integrating KRT7 protein expression analysis with RNA-seq data provides a comprehensive understanding of epithelial cancer biology. This multi-omics approach reveals relationships between transcriptional and translational regulation of KRT7.

Methodological approach:

• Perform immunohistochemistry with KRT7 antibodies on cancer tissues and matched normal samples to quantify protein expression patterns.

• Conduct parallel RNA-seq to measure KRT7 mRNA levels in the same samples.

• Analyze correlation between KRT7 protein and mRNA levels to identify potential post-transcriptional regulation mechanisms.

• Examine the relationship between KRT7 and KRT7-AS (antisense) RNA expression, as KRT7-AS has been shown to reduce oncogenic KRT7 levels in cancer cells .

• Conduct pathway analysis incorporating KRT7-associated genes identified through RNA-seq.

Research has demonstrated that KRT7 mRNA levels can be increased by sevenfold and ninefold in lung and breast tumors, respectively, while KRT7-AS levels are significantly reduced in these cancers . This integrated approach can reveal whether discrepancies between mRNA and protein levels might be explained by antisense RNA regulation or other post-transcriptional mechanisms.

What approaches can researchers use to investigate the relationship between KRT7 and KRT7-AS in cancer progression?

Investigating the relationship between KRT7 and its antisense RNA (KRT7-AS) in cancer progression requires multiple experimental approaches:

• Expression correlation studies:

  • Quantify KRT7 and KRT7-AS levels using qPCR and Western blot in paired tumor/normal tissues

  • Conduct statistical analysis to determine inverse correlation patterns

  • Research has demonstrated that KRT7-AS expression is significantly lower in nine cancer types compared to matched normal tissues, with a 5.3-fold reduction in lung cancer samples

• Mechanistic investigations:

  • Perform overexpression and knockdown experiments of KRT7-AS to observe effects on KRT7 protein levels

  • Use RNA immunoprecipitation to confirm direct interaction between KRT7-AS and KRT7 mRNA

  • The complementary binding region between KRT7-AS and KRT7 mRNA spans 213 nucleotides with 100% complementarity

• Functional studies:

  • Evaluate cancer cell phenotypes (proliferation, migration, apoptosis) after modulating KRT7-AS levels

  • Assess drug sensitivity changes - KRT7-AS overexpression significantly increases cancer cell sensitivity to cisplatin

  • Analyze downstream effectors like PTEN and FOXA1, as KRT7-AS has been shown to elevate the tumor suppressor PTEN while reducing oncogenic FOXA1 levels

• Clinical correlation analysis:

  • Evaluate patient survival based on KRT7/KRT7-AS expression ratio

  • Patients with low KRT7-AS levels show significantly reduced survival compared to those with high KRT7-AS expression

These approaches provide comprehensive insights into the regulatory dynamics between KRT7 and KRT7-AS in cancer biology.

How can researchers design experiments to evaluate KRT7's role in drug resistance mechanisms?

Designing experiments to evaluate KRT7's role in drug resistance requires a systematic approach focusing on both mechanistic understanding and clinical relevance:

• Expression correlation with drug response:

  • Quantify KRT7 expression across cancer cell lines with varying drug sensitivity profiles

  • Analyze public datasets (GDSC, CCLE) for correlations between KRT7 expression and drug response patterns

  • Compare KRT7 levels in matched pre- and post-treatment patient samples

• Genetic manipulation experiments:

  • Generate stable KRT7 overexpression and knockdown models in relevant cancer cell lines

  • Perform drug sensitivity assays using clinically relevant compounds (e.g., cisplatin)

  • Research shows that KRT7-AS (which reduces KRT7 levels) significantly increases cancer cell sensitivity to cisplatin by enhancing apoptosis five-fold in lung cancer cells

• Mechanistic pathway analysis:

  • Investigate how KRT7 modulation affects established drug resistance pathways:

    • Apoptosis machinery (measure activation of caspases, Bcl-2 family proteins)

    • DNA damage repair mechanisms

    • Drug efflux pump expression and activity

    • PTEN tumor suppressor levels, as KRT7-AS increases PTEN while reducing KRT7

• Combination strategy evaluation:

  • Test whether KRT7 targeting (via siRNA/shRNA) enhances conventional chemotherapy efficacy

  • Investigate whether modulating KRT7-AS levels alters drug response

  • Design time-course experiments to determine optimal sequencing of KRT7 targeting and drug administration

• Validation in advanced models:

  • Confirm findings in 3D organoid cultures and patient-derived xenograft models

  • Correlate results with patient treatment outcomes when possible

This comprehensive experimental design helps elucidate KRT7's specific contributions to drug resistance mechanisms while identifying potential therapeutic vulnerabilities.

How should researchers analyze KRT7 expression patterns across different tissue types?

Analyzing KRT7 expression patterns across tissue types requires a structured approach combining quantitative and qualitative methodologies:

• Tissue microarray (TMA) analysis:

  • Create or obtain TMAs containing diverse normal and pathological tissues

  • Perform immunohistochemistry using validated KRT7 antibodies at standardized dilutions (1:20-1:200)

  • Develop a scoring system incorporating:

    • Percentage of KRT7-positive cells (0-100%)

    • Staining intensity (0-3+ scale)

    • Subcellular localization patterns (cytoplasmic, membranous)

  • Calculate H-scores (0-300) by multiplying intensity by percentage

• Comparative tissue expression profiling:

  • Systematically document KRT7 expression across epithelial tissues (bladder, gastrointestinal tract, bile ducts)

  • Create expression heat maps highlighting tissue-specific patterns

  • Correlate with other epithelial markers to identify co-expression patterns

• Pathological context analysis:

  • Compare KRT7 expression between normal and neoplastic tissues of the same origin

  • Analyze expression changes during disease progression

  • Research shows KRT7 is significantly overexpressed in lung and breast tumor tissues compared to adjacent normal tissues

• Integrated multi-omics analysis:

  • Correlate protein expression with mRNA levels across tissues

  • Investigate regulatory mechanisms including antisense RNA (KRT7-AS) that may explain tissue-specific expression differences

  • Examine relationships with other molecular characteristics (mutation profiles, pathway activation)

This comprehensive approach provides insights into both physiological and pathological KRT7 expression patterns, facilitating more accurate interpretation of experimental results.

What statistical approaches are most appropriate for analyzing KRT7 expression in cancer versus normal tissues?

When analyzing KRT7 expression differences between cancer and normal tissues, researchers should employ rigorous statistical approaches tailored to the specific data characteristics:

• For continuous expression data (qPCR, Western blot densitometry):

  • Paired t-test for matched tumor-normal samples from the same patient

  • Welch's t-test for unpaired samples with potentially unequal variance

  • Mann-Whitney U test for non-normally distributed data

  • Research demonstrates that KRT7 mRNA levels were increased by sevenfold in lung tumors and ninefold in breast tumors compared to normal tissues

• For semi-quantitative IHC scoring:

  • Wilcoxon signed-rank test for paired ordinal data

  • Chi-square or Fisher's exact test for categorical expression levels

  • Cohen's kappa to assess inter-observer agreement on scoring

• For large-scale multi-cohort analysis:

  • Meta-analysis approaches to combine effect sizes across studies

  • Random-effects models to account for between-study heterogeneity

  • Forest plots to visualize expression differences across cancer types

  • In a large-scale analysis of 687 lung cancer samples, KRT7-AS expression was reduced 5.3-fold compared to adjacent normal tissues (p<0.001)

• For survival analysis:

  • Kaplan-Meier curves with log-rank tests to compare survival between high/low KRT7 expression groups

  • Cox proportional hazards models to adjust for clinical covariates

  • Time-dependent ROC curve analysis to evaluate KRT7's prognostic value

  • Research shows patients with low KRT7-AS levels (which correlates with high KRT7) had significantly reduced survival times

• For experimental design considerations:

  • Power analysis to determine adequate sample sizes

  • Multiple testing correction (Bonferroni, FDR) for genome-wide studies

  • Randomization and blinding protocols to reduce experimental bias

These statistical approaches ensure robust analysis of KRT7 expression differences while accounting for biological and technical variability.

How can researchers reconcile contradictory findings about KRT7's role in different cancer types?

Reconciling contradictory findings about KRT7's role across different cancer types requires a systematic analytical framework:

• Contextual analysis of molecular interactions:

  • Investigate cancer-specific co-expression patterns with KRT7

  • Analyze tissue-specific regulatory mechanisms

  • Examine interactions with KRT7-AS, which has divergent roles in different cancers:

    • Enhances tumorigenesis in gastric and colorectal cancers by stabilizing KRT7 mRNA

    • Forms a duplex with KRT7 that promotes lung metastasis in breast cancer

    • Acts as a tumor suppressor in lung cancer by reducing KRT7 levels

• Methodological harmonization and critical evaluation:

  • Compare experimental approaches across contradictory studies

  • Evaluate antibody specificity and validation methods

  • Assess cell line authenticity and relevance to cancer subtypes

  • Consider differences in:

    • In vitro vs. in vivo models

    • 2D vs. 3D culture systems

    • Genetic manipulation techniques

• Multi-omics integration:

  • Correlate KRT7 protein expression with:

    • Genetic alterations in the KRT7 gene locus

    • Epigenetic modifications affecting KRT7 regulation

    • Transcriptional programs specific to cancer subtypes

  • Investigate post-translational modifications that might alter KRT7 function

• Pathway-focused analysis:

  • Map KRT7 involvement in context-dependent signaling networks

  • Identify cancer-specific downstream effectors

  • Research shows KRT7 can influence different downstream factors:

    • Oncogenic FOXA1 in some contexts

    • Tumor suppressor PTEN in others

• Systematic hypothesis generation:

  • Develop testable models explaining context-dependent functions

  • Design experiments to directly compare KRT7 functions across cancer types under identical conditions

  • Use CRISPR-Cas9 screening to identify synthetic lethal interactions specific to each cancer type

This comprehensive approach helps reconcile apparently contradictory findings by revealing cancer-specific molecular contexts that modify KRT7's functional impact.

What are promising research areas for investigating KRT7-AS as a therapeutic target in cancer?

Investigating KRT7-AS as a therapeutic target presents several promising research directions based on its tumor-suppressive properties:

• RNA-based therapeutic development:

  • Design synthetic KRT7-AS mimics for cancer therapy

  • Develop targeted delivery systems (nanoparticles, lipid carriers) to increase KRT7-AS levels in tumor tissues

  • Investigate optimization of RNA stability and cellular uptake

  • Evaluate combination approaches with conventional chemotherapeutics, as KRT7-AS overexpression significantly increases cancer cell sensitivity to cisplatin

• Mechanistic understanding for drug development:

  • Further characterize the 213-nucleotide complementary binding region between KRT7-AS and KRT7 mRNA

  • Identify the minimal functional sequence required for KRT7 regulation

  • Design small molecule compounds that stabilize KRT7-AS/KRT7 mRNA interactions

  • Investigate structure-based design of molecular mimics

• Pathway modulation strategies:

  • Explore approaches to modulate KRT7-AS-mediated elevation of PTEN tumor suppressor

  • Develop therapeutic strategies targeting the KRT7/FOXA1 oncogenic axis

  • Investigate synergistic combinations with existing PTEN pathway modulators

  • Screen for compounds that selectively upregulate KRT7-AS expression

• Predictive biomarker development:

  • Develop assays to measure KRT7-AS/KRT7 ratio as a predictive biomarker for therapy response

  • Identify patient subgroups most likely to benefit from KRT7-AS-targeted approaches

  • Research shows patients with low KRT7-AS levels show significantly reduced survival times

  • Validate these biomarkers in retrospective and prospective clinical studies

• Translation to clinical applications:

  • Develop clinically applicable delivery methods for RNA therapeutics targeting epithelial cancers

  • Design rational combination strategies with immunotherapy, targeted therapy, and conventional chemotherapy

  • Establish appropriate patient selection criteria based on molecular profiling

These research directions leverage the tumor-suppressive properties of KRT7-AS while addressing practical challenges in therapeutic development and clinical translation.

How might researchers design experiments to investigate the role of KRT7 in cancer stemness and metastasis?

Designing experiments to investigate KRT7's role in cancer stemness and metastasis requires sophisticated approaches across multiple model systems:

• Cancer stem cell (CSC) characterization:

  • Isolate putative CSC populations using established markers (CD44+/CD24-, ALDH+, etc.)

  • Quantify KRT7 expression in CSC versus non-CSC populations

  • Perform functional assays (sphere formation, serial transplantation) after KRT7 modulation

  • Analyze correlation between KRT7 and stemness-related transcription factors

• Lineage tracing experiments:

  • Generate reporter systems driven by the KRT7 promoter

  • Track the fate of KRT7-expressing cells during tumor progression

  • Use inducible systems to temporally control KRT7 expression

  • Correlate with acquisition of stem-like properties and metastatic potential

• 3D organoid and patient-derived xenograft (PDX) models:

  • Establish organoids from primary tumors with varying KRT7 expression

  • Manipulate KRT7 levels using CRISPR-Cas9 or shRNA approaches

  • Assess organoid-forming efficiency, differentiation capacity, and drug resistance

  • Evaluate tumorigenicity and metastatic potential in PDX models

• Metastasis models and analysis:

  • Utilize spontaneous and experimental metastasis assays (tail vein, intracardiac injection)

  • Perform intravital imaging to track KRT7-expressing cells during metastatic spread

  • Analyze circulating tumor cells (CTCs) for KRT7 expression

  • Research indicates KRT7-AS and KRT7 duplex formation can promote lung metastasis in breast cancer

• Mechanistic pathway investigation:

  • Conduct RNA-seq and proteomics after KRT7 modulation to identify stemness and metastasis pathways

  • Investigate epithelial-mesenchymal transition (EMT) markers in relation to KRT7 expression

  • Examine influence on tumor microenvironment and immune evasion

  • Analyze relationship with PTEN tumor suppressor, as KRT7-AS (which reduces KRT7) increases PTEN levels

• Clinical correlation studies:

  • Analyze KRT7 expression patterns at invasive fronts versus tumor centers

  • Correlate KRT7 levels with metastatic burden and patterns

  • Examine KRT7 expression in paired primary and metastatic lesions

These experimental approaches provide comprehensive insights into KRT7's contributions to cancer stemness and metastasis across multiple model systems.

What emerging technologies could enhance KRT7-related cancer research?

Several emerging technologies offer significant potential to advance KRT7-related cancer research:

• Spatial transcriptomics and proteomics:

  • Map KRT7 and KRT7-AS expression patterns with spatial resolution in tumor microenvironments

  • Correlate with other cancer markers to identify spatial heterogeneity

  • Visualize KRT7 expression at tumor-stroma interfaces and invasive fronts

  • Integrate with multiplexed immunofluorescence to simultaneously detect multiple proteins

• Single-cell multi-omics:

  • Perform single-cell RNA-seq to identify cell populations with distinct KRT7 expression profiles

  • Combine with single-cell ATAC-seq to understand chromatin accessibility at the KRT7 locus

  • Implement single-cell proteomics to correlate KRT7 protein levels with cellular phenotypes

  • Identify rare cell populations that might drive KRT7-associated tumor behaviors

• CRISPR screening technologies:

  • Conduct genome-wide CRISPR screens to identify synthetic lethal interactions with KRT7

  • Use CRISPRa/CRISPRi libraries to identify regulators of KRT7 and KRT7-AS expression

  • Perform CRISPR base editing to study the impact of specific KRT7 mutations

  • Implement CRISPR-based lineage tracing to track KRT7-expressing cells during tumor evolution

• Advanced 3D and in vivo models:

  • Develop patient-derived tumor organoids with controlled KRT7 expression

  • Create microfluidic organ-on-chip models incorporating KRT7-expressing cancer cells

  • Implement bioprinting technologies to generate complex 3D tumor models

  • Utilize humanized mouse models to study KRT7's role in tumor-immune interactions

• RNA-targeted therapeutics development platforms:

  • Design antisense oligonucleotides to modulate KRT7/KRT7-AS balance

  • Develop small molecule RNA-binding molecules targeting the KRT7-AS/KRT7 complementary binding region

  • Create nanoparticle delivery systems for tissue-specific RNA therapeutic delivery

  • Utilize high-throughput screening to identify compounds that selectively modulate KRT7-AS expression

• Artificial intelligence and machine learning:

  • Apply deep learning to analyze KRT7 staining patterns in histopathology

  • Develop predictive models for patient outcomes based on KRT7 expression signatures

  • Utilize AI for drug discovery targeting KRT7-related pathways

  • Implement natural language processing to synthesize findings across KRT7 literature

These technologies will significantly accelerate understanding of KRT7's complex roles in cancer biology while facilitating translation to clinical applications.