KRT7 Antibody

What is KRT7 and where is it expressed in normal tissues?

KRT7 (Keratin 7 or Cytokeratin 7) is a type II cytokeratin protein with a molecular weight of approximately 51kDa that functions as an intermediate filament protein in epithelial cells . It's primarily expressed in glandular and transitional epithelia but not in stratified squamous epithelia . Specifically, KRT7 is found in epithelial cells of the ovary, lung, and breast, but is notably absent in the colon, prostate, and gastrointestinal tract . This differential expression pattern makes KRT7 a valuable marker for distinguishing between different types of epithelial tissues in research and diagnostic applications.

What applications are KRT7 antibodies commonly used for in research?

KRT7 antibodies are utilized across multiple experimental platforms including:

• Western blotting (WB) for protein expression quantification

• Immunohistochemistry on paraffin-embedded tissues (IHC-P) for localization studies

• Immunocytochemistry/Immunofluorescence (ICC/IF) for cellular distribution analysis

• Flow cytometry for cell population characterization

• ELISA for protein quantification in solution

The versatility of these applications makes KRT7 antibodies valuable tools for both epithelial biology research and cancer studies.

How do I optimize IHC protocols when using KRT7 antibodies?

For optimal immunohistochemistry results with KRT7 antibodies:

• Use formalin-fixed, paraffin-embedded tissues with recommended antibody concentrations (typically 1-2 μg/ml)

• Perform heat-induced epitope retrieval by heating tissue sections in 10mM Tris Buffer with 1mM EDTA, pH 9.0, for 45 minutes at 95°C, followed by cooling at room temperature for 20 minutes

• Incubate with primary antibody for approximately 30 minutes at room temperature

• Use appropriate detection systems based on host species (typically mouse or rabbit)

• Include positive control tissues known to express KRT7 (such as lung or ovarian tissues)

How is KRT7 expression altered in different cancer types?

Research shows distinct KRT7 expression patterns across various malignancies:

This differential expression pattern makes KRT7 valuable for both diagnostic applications and prognostic assessments in cancer research .

What mechanisms regulate KRT7 expression in cancer cells?

The regulation of KRT7 in cancer involves multiple mechanisms:

• Post-transcriptional regulation: The long non-coding RNA KRT7-AS (antisense) forms a 213-nucleotide complementary sequence with KRT7 mRNA, which typically reduces KRT7 protein levels . In many cancers, KRT7-AS is downregulated, resulting in elevated KRT7 expression .

• RNA stability modulation: RNA-binding proteins like IGF2BP3 can enhance KRT7 mRNA stability. In pancreatic cancer, IGF2BP3 expression correlates strongly with KRT7 (correlation coefficient 0.63-0.66), and silencing IGF2BP3 decreases KRT7 protein levels .

• Transcriptional control: Altered transcription factor activity in cancer cells can lead to aberrant KRT7 expression, with downstream effects on oncogenic pathways including FOXA1 activation .

Understanding these regulatory mechanisms provides potential therapeutic targets for cancers with aberrant KRT7 expression.

What is the relationship between KRT7 and tumor metastasis?

KRT7 plays significant roles in promoting cancer metastasis, particularly in pancreatic cancer:

• In animal models, KRT7-overexpressing PANC-1 cells injected into immunodeficient mice showed significantly increased lung metastases compared to control cells .

• Mechanistically, KRT7 appears to influence pathways related to mesenchymal-to-epithelial transition, which is critical for metastatic colonization .

• The relationship between KRT7 and other oncogenic proteins like FOXA1 contributes to enhanced metastatic potential in cancer cells .

These findings establish KRT7 as not merely a diagnostic marker but as a functional contributor to the metastatic process, highlighting its potential as a therapeutic target in aggressive cancers.

How do I select the appropriate KRT7 antibody for my specific research application?

When selecting a KRT7 antibody, consider these critical parameters:

• Antibody format: Monoclonal antibodies (such as KRT7/760, KRT7/903, KRT7/1499R) offer high specificity and reproducibility for applications requiring precise epitope recognition . Polyclonal antibodies provide broader epitope coverage but may introduce batch-to-batch variability .

• Host species: Consider the host species (rabbit, mouse) in relation to your experimental design, particularly for co-staining experiments to avoid cross-reactivity .

• Validated applications: Confirm the antibody has been validated specifically for your intended application (WB, IHC-P, IF/ICC, Flow Cytometry) .

• Epitope information: The specific region of KRT7 recognized by the antibody can impact detection in different experimental contexts. Some antibodies target the full-length protein while others recognize specific domains .

• Reactivity with post-translational modifications: If your research focuses on modified forms of KRT7, ensure the antibody's epitope is not affected by relevant modifications.

What are the best practices for troubleshooting KRT7 detection in Western blot applications?

When troubleshooting KRT7 detection by Western blot:

• Sample preparation: KRT7 is cytoskeletal and may require specialized lysis buffers containing sufficient detergent (e.g., RIPA buffer with 1% SDS) to fully solubilize the protein.

• Expected molecular weight: Confirm bands at approximately 51kDa, the reported molecular weight for KRT7 . Be aware that post-translational modifications may cause shifts in migration patterns.

• Positive controls: Include lysates from cell lines known to express KRT7 (e.g., HeLa cells, lung epithelial cells) as positive controls .

• Blocking optimization: For recalcitrant antibodies, test alternative blocking agents (BSA vs. non-fat milk) as certain antibodies may be sensitive to blocking conditions.

• Signal enhancement: For low abundance samples, consider signal amplification systems or increased antibody concentration with extended incubation times.

• Cross-reactivity assessment: Ensure specificity by confirming the absence of bands in KRT7-negative tissues (e.g., colon epithelium) to exclude cross-reactivity with other cytokeratins.

How can I quantitatively assess KRT7 expression across different experimental conditions?

For rigorous quantitative analysis of KRT7 expression:

• Western blot quantification:

  • Use appropriate normalization controls (GAPDH, β-actin, total protein staining)

  • Employ digital image analysis software to measure band intensity

  • Ensure signal is within linear range of detection

  • Run technical replicates (n≥3) for statistical validity

• qRT-PCR for mRNA expression:

  • Design primers specific to KRT7 (avoiding cross-detection of other keratins)

  • Use validated reference genes appropriate for your experimental system

  • Apply the 2^-ΔΔCT method for relative quantification

  • Include mRNA stability assessments when studying regulatory mechanisms

• Immunofluorescence quantification:

  • Use consistent acquisition parameters (exposure time, gain)

  • Quantify signal intensity across multiple fields and cells

  • Apply appropriate background correction

  • Consider subcellular distribution (primarily cytoplasmic for KRT7)

How does KRT7-AS function as a tumor suppressor through regulation of KRT7?

The long non-coding RNA KRT7-AS functions as a tumor suppressor through multiple mechanisms:

• Direct regulation of KRT7: KRT7-AS forms a complementary duplex with KRT7 mRNA through a 213-nucleotide region with 100% complementarity, which typically leads to degradation of KRT7 mRNA. In cancer cells where KRT7-AS is downregulated, KRT7 protein levels increase, promoting tumorigenesis .

• PTEN modulation: Surprisingly, KRT7-AS increases levels of the tumor suppressor PTEN. This effect enhances apoptotic sensitivity and reduces cancer cell survival, particularly in response to chemotherapeutic agents like cisplatin .

• DNA damage response enhancement: KRT7-AS overexpression increases levels of γ-H2AX, a marker of DNA strand breaks, especially after cisplatin treatment. This suggests KRT7-AS sensitizes cancer cells to DNA-damaging therapies .

• Downstream pathway regulation: KRT7-AS suppresses oncogenic pathways including FOXA1, which is elevated in many cancers with low KRT7-AS expression .

These mechanisms establish KRT7-AS as a potential therapeutic target, where restoration of its expression might sensitize resistant tumors to conventional treatments.

What methodological approaches can detect the interaction between KRT7 and KRT7-AS?

To investigate KRT7/KRT7-AS interactions, researchers can employ these specialized techniques:

• RNA Fluorescence in situ Hybridization (FISH): Enables visualization of KRT7-AS localization within cells, confirming its predominantly cytoplasmic distribution where it can interact with KRT7 mRNA .

• RNA immunoprecipitation (RIP): Allows isolation of KRT7 mRNA-protein complexes that may include KRT7-AS, providing evidence of physical interactions in cellular contexts.

• RNA stability assays: Using transcription inhibitors like Actinomycin D, researchers can measure KRT7 mRNA half-life in the presence or absence of KRT7-AS to determine if the interaction affects mRNA stability .

• Dual luciferase reporter assays: By cloning the complementary region of KRT7 mRNA into a reporter construct, researchers can directly measure the impact of KRT7-AS on expression.

• RNA pulldown assays: Using biotinylated KRT7-AS transcripts to capture interacting partners, followed by mass spectrometry, to identify proteins involved in the KRT7/KRT7-AS regulatory complex.

How can KRT7 expression analysis be integrated into cancer prognostic models?

KRT7 serves as a valuable prognostic biomarker that can be integrated into clinical models through:

• Multivariate risk scoring systems: In pancreatic cancer, KRT7 has been identified as the most significant risk gene (coefficient = 0.19) in prognostic models that stratify patients into high-risk and low-risk groups .

• Survival analysis integration: Kaplan-Meier survival analysis comparing high versus low KRT7 expression demonstrates significant prognostic value across multiple cancer datasets, including GSE28735, GSE62452, and GSE71729 .

• ROC curve validation: Area Under the Curve (AUC) values for KRT7-based models show strong predictive performance: 1- and 3-year AUC values of 0.593 and 0.825 in GSE28735; 1-, 3-, and 5-year AUC values of 0.628, 0.879, and 0.911 in GSE62452 .

• Multiparameter tissue analysis: Combining KRT7 expression with other molecular markers (like IGF2BP3) significantly enhances prognostic accuracy beyond single-marker approaches .

• Methodological standardization: For clinical implementation, standardized immunohistochemical scoring systems for KRT7 must be established to ensure reproducibility across laboratories and patient cohorts.

What therapeutic opportunities exist for targeting the KRT7/KRT7-AS axis in cancer?

The KRT7/KRT7-AS regulatory axis presents several promising therapeutic approaches:

• KRT7-AS restoration therapy: Since KRT7-AS functions as a tumor suppressor and is downregulated in multiple cancers, delivery systems that restore KRT7-AS expression could suppress oncogenic KRT7 and enhance chemosensitivity .

• Small molecule modulators: Compounds that disrupt the interaction between IGF2BP3 and KRT7 mRNA could reduce KRT7 stability and expression, potentially inhibiting tumor progression and metastasis .

• Combination approaches: KRT7-AS restoration sensitizes cancer cells to cisplatin by enhancing DNA damage responses, suggesting potential synergistic effects with conventional chemotherapies .

• PTEN pathway enhancement: Since KRT7-AS increases PTEN levels, therapies targeting this axis might restore tumor suppression through multiple downstream pathways beyond just KRT7 regulation .

• Metastasis inhibition: Given KRT7's role in promoting metastasis, particularly in pancreatic cancer, interventions targeting KRT7 might specifically reduce metastatic potential even in established tumors .

These therapeutic strategies remain in preclinical stages but represent promising directions for translational cancer research.

How do post-translational modifications affect KRT7 function in different cellular contexts?

Post-translational modifications (PTMs) of KRT7 represent an understudied but potentially critical aspect of its function:

• Phosphorylation: While not extensively characterized for KRT7 specifically, phosphorylation of keratins generally regulates their assembly/disassembly dynamics and interactions with signaling proteins during stress responses and mitosis.

• Glycosylation: Keratin glycosylation can alter structural properties and interactions with other cellular components, potentially affecting epithelial cell function in normal and pathological states.

• Ubiquitination: This modification likely regulates KRT7 turnover and may be dysregulated in cancer cells, contributing to aberrant KRT7 accumulation.

• Acetylation: Histone deacetylase inhibitors have been shown to affect keratin expression and organization, suggesting acetylation may regulate KRT7 function.

• PTM detection methods: Specialized techniques including mass spectrometry, phospho-specific antibodies, and PTM-focused proteomics are required to fully characterize the KRT7 "PTM code" across different cellular contexts.

This represents an emerging area where additional research could reveal new regulatory mechanisms and therapeutic opportunities.

What are the most reliable cell line models for studying KRT7 biology?

Based on the available research, these cell line models show consistent KRT7 expression patterns:

When selecting models, researchers should verify baseline KRT7 expression in their specific cell line stocks, as expression can vary with passage number and culture conditions.

What experimental design considerations are important when studying KRT7-AS effects on cancer cells?

When investigating KRT7-AS function, critical experimental design factors include:

• Expression validation: Confirm KRT7-AS expression levels using qRT-PCR before functional assays, as baseline expression varies significantly across cell lines .

• Cellular localization: Verify cytoplasmic localization of KRT7-AS using RNA FISH, as this is critical for its interaction with KRT7 mRNA .

• Gain/loss-of-function approaches:

  • For overexpression: Use vector systems with strong promoters (CMV) for robust expression

  • For silencing: Test multiple siRNA/shRNA constructs targeting different regions of KRT7-AS

  • Include rescue experiments to confirm specificity of observed phenotypes

• Functional readouts:

  • Colony formation assays for tumorigenicity assessment

  • Cisplatin sensitivity testing at multiple concentrations

  • Apoptosis markers including γ-H2AX immunostaining

  • Western blot for KRT7 and downstream effectors like PTEN

• Temporal considerations: Monitor effects over time, as some phenotypes (like enhanced cisplatin sensitivity) show time-dependent characteristics .

• Controls: Include proper vector-only controls for overexpression studies and non-targeting siRNA/shRNA controls for silencing experiments.