KRT1 Monoclonal Antibody

What is Keratin 1 (KRT1) and why is it significant in epithelial research?

Keratin 1 belongs to the family of intermediate filament proteins that form heterodimers composed of type I keratins (9-23) and type II keratins (1-8). It is a differentiation-specific keratin that predominates in suprabasal keratinocytes of stratified epithelia . KRT1's significance in epithelial research stems from its essential role in maintaining epithelial integrity, with mutations in the KRT1 gene causing epidermolytic hyperkeratosis . Additionally, KRT1 regulates cellular processes including proliferation and apoptosis, as evidenced by experiments showing dose-dependent inhibition of cell proliferation when treated with KRT1 antibody . For researchers investigating epithelial biology, KRT1 serves as a marker for differentiated keratinocytes and plays a critical role in barrier function.

What are the standard applications for KRT1 monoclonal antibodies in research?

KRT1 monoclonal antibodies are utilized in multiple experimental applications, with immunohistochemistry (IHC) and ELISA being the most common . For IHC applications, the recommended dilution ranges from 1:20 to 1:200, depending on tissue type and fixation methods . These antibodies are valuable tools for detecting KRT1 expression in human tissue samples, particularly in stratified epithelia where KRT1 is abundantly expressed . Additionally, KRT1 antibodies can be employed in cellular assays to assess the regulatory roles of KRT1 in cell proliferation and apoptosis, as demonstrated in Caco-2 cell studies where concentration gradients (1, 5, and 10 ng/mL) revealed dose-dependent effects on cellular functions .

How do I select the appropriate conjugated KRT1 antibody for my fluorescence imaging experiments?

Selection of the appropriate conjugated KRT1 antibody depends on your experimental setup, including microscopy equipment, fluorescent proteins already in use, and target abundance. KRT1 antibodies are available with various fluorescent CF® dye conjugations that offer exceptional brightness and photostability . The selection should consider:

It's important to note that conjugates of blue fluorescent dyes (CF®405S) are not recommended for detecting low-abundance targets due to lower fluorescence and potentially higher non-specific background compared to other dye colors . For multiplexing experiments, choose conjugations with minimal spectral overlap with other fluorophores in your experimental design.

How does KRT1 modulate intestinal barrier function in inflammatory conditions such as ulcerative colitis?

KRT1 plays a complex role in maintaining intestinal barrier integrity through multiple mechanisms. Recent research demonstrates that KRT1 enhances the expression of tight junction proteins including occludin, zonula occludens-1 (ZO-1), and claudin, which are critical components of the intestinal mechanical barrier . In ulcerative colitis models, KRT1 treatment significantly upregulated these tight junction proteins at both mRNA and protein levels (P < 0.001), contributing to barrier repair and reduced intestinal permeability .

KRT1 also modulates intestinal barrier function through regulation of the kallikrein kinin system (KKS). Studies show that KRT1 inhibits the expression of coagulation factor XIIα, a negative regulator of intestinal barrier function . Additionally, KRT1 suppresses bradykinin (BK) expression and modulates high molecular weight kininogen (HK) levels, thereby attenuating KKS-mediated inflammation . This multi-faceted approach to barrier regulation makes KRT1 a promising therapeutic target for inflammatory bowel diseases.

Methodologically, researchers investigating KRT1's role in intestinal inflammation should consider both in vitro approaches using intestinal epithelial cell lines (e.g., Caco-2) and in vivo models such as dextran sulfate sodium (DSS)-induced colitis in mice, which has demonstrated KRT1's ability to alleviate colonic injury and promote barrier repair .

What are the molecular interactions between KRT1 and inflammatory signaling pathways?

KRT1's interactions with inflammatory signaling pathways are multifaceted and context-dependent. At the molecular level, KRT1 may regulate the activity of protein kinases such as PKC and SRC via binding to integrin beta-1 (ITB1) and the receptor of activated protein C kinase 1 (RACK1) . This interaction influences downstream signaling events that modulate inflammatory responses.

In inflammatory conditions like ulcerative colitis, KRT1 significantly inhibits the activation of the nuclear factor kappa-B signaling pathway, consequently reducing the production of pro-inflammatory cytokines including tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and interleukin-6 (IL-6) . Experimental evidence from DSS-induced mouse models demonstrates that KRT1 treatment significantly suppresses these inflammatory cytokines by blocking bradykinin activation .

Additionally, KRT1 forms a complex with C1QBP, creating a high-affinity receptor for kininogen-1/HMWK, which further influences inflammatory cascades . The interaction between KRT1 and the Wnt/β-catenin signaling pathway also promotes regeneration and repair of intestinal epithelial cells, enhancing barrier function and alleviating inflammation .

For researchers investigating these interactions, immunoprecipitation assays, protein-protein interaction studies, and signaling pathway analyses using phospho-specific antibodies provide valuable methodological approaches.

How can I optimize KRT1 antibody concentration for studying its effects on cell proliferation and apoptosis?

Optimizing KRT1 antibody concentration requires a systematic approach based on the specific cell type and experimental endpoints being measured. Based on recent studies with Caco-2 cells, a concentration gradient test approach is recommended, starting with concentrations of 1, 5, and 10 ng/mL .

Research has demonstrated that KRT1 antibody inhibits cellular proliferation in a dose-dependent manner, with greater suppression evident at higher concentrations . For apoptosis studies, it's important to note that concentration thresholds may exist - at lower concentrations (1 ng/mL), KRT1 antibody showed no significant effect on cellular apoptosis, while concentrations exceeding 10 ng/mL significantly enhanced apoptosis (P < 0.001) .

The optimal experimental design should include:

• A preliminary concentration gradient study spanning at least 3 log orders (e.g., 0.1, 1, 10, 100 ng/mL)

• Multiple timepoints (24, 48, 72 hours) to capture temporal dynamics

• Both proliferation assays (MTT or BrdU incorporation) and apoptosis measurements (Annexin V/PI staining or caspase activity)

• Appropriate controls including isotype-matched control antibodies

This approach provides a comprehensive analysis of concentration-dependent effects while establishing the threshold concentrations for specific cellular responses.

What techniques are most effective for assessing KRT1-mediated changes in epithelial barrier function?

Assessing KRT1-mediated changes in epithelial barrier function requires a multi-parametric approach combining molecular, cellular, and functional analyses. Based on research protocols used to evaluate KRT1's role in intestinal barrier function, the following methodologies are recommended:

• Tight Junction Protein Analysis: Use RT-qPCR and Western blotting to quantify expression levels of key barrier proteins including occludin, ZO-1, and claudin at both mRNA and protein levels . Immunofluorescence microscopy provides additional spatial information about tight junction distribution.

• Trans-epithelial Electrical Resistance (TEER): Measure epithelial monolayer integrity in real-time using electrode-based systems to detect subtle changes in barrier function following KRT1 antibody treatment or KRT1 protein administration.

• Paracellular Permeability Assays: Employ fluorescently-labeled dextrans of various molecular weights to assess size-selective barrier properties, which can reveal mechanism-specific alterations in paracellular transport.

• In Vivo Barrier Assessment: For translational studies, combine histological assessment (H&E staining) with localized permeability tests using orally administered FITC-dextran followed by serum fluorescence quantification .

• Inflammatory Marker Profiling: Quantify inflammatory cytokines (IL-1, IL-6, TNF-α) using ELISA to correlate barrier disruption with inflammatory status .

These complementary approaches provide a comprehensive assessment of barrier function that captures both molecular alterations and functional outcomes of KRT1 modulation.

How should I design experimental controls when studying KRT1 antibody effects in cellular and animal models?

Designing appropriate controls is critical for robust interpretation of KRT1 antibody effects. Based on established experimental approaches, the following control strategies are recommended:

For cellular models:

• Isotype Controls: Include isotype-matched antibodies (e.g., mouse IgG1 for KRT1 monoclonal antibodies) at equivalent concentrations to control for non-specific effects .

• Concentration Gradients: Implement multiple antibody concentrations (e.g., 1, 5, 10 ng/mL) to establish dose-response relationships and identify threshold effects .

• Vehicle Controls: Include all buffer components without antibody to account for potential carrier effects.

• Positive Controls: For studies involving barrier function, include known barrier disruptors (e.g., EGTA, cytokines) as positive controls.

• siRNA Validation: Complement antibody studies with KRT1 knockdown experiments to confirm specificity.

For animal models:

• Control Groups: Include both negative controls (untreated) and positive controls (e.g., DSS-only groups in colitis models) .

• Antibody Controls: Administer isotype-matched non-specific antibodies to control animals.

• Intervention Timing: Establish both preventive and therapeutic administration protocols to distinguish between protective and restorative effects.

• Recovery Groups: Include groups allowed to recover after intervention cessation to assess long-term effects.

These control strategies ensure experimental rigor and facilitate interpretation of KRT1-specific effects versus non-specific or secondary phenomena.

What are the best approaches for analyzing KRT1's interactions with the kallikrein kinin system components?

Analyzing KRT1's interactions with the kallikrein kinin system (KKS) requires specialized methodologies focusing on both expression and functional analyses. Based on recent research investigating KRT1-KKS interactions in inflammatory conditions, the following approaches are recommended:

• Expression Analysis of KKS Components: Employ immunofluorescence and ELISA to quantify high molecular weight kininogen (HK) and bradykinin (BK) levels in response to KRT1 antibody treatment at various concentrations . This approach demonstrated that KRT1 antibody reduces HK expression while increasing BK production in Caco-2 cells.

• Coagulation Factor XIIα Assessment: Measure factor XIIα activity and expression using activity assays and Western blotting to evaluate KRT1's regulatory effect on this intestinal barrier factor . Research has shown that KRT1 antibody significantly upregulates FXIIα, impacting barrier function.

• Protein-Protein Interaction Studies: Implement co-immunoprecipitation assays to investigate direct interactions between KRT1 and C1QBP, which together form a high-affinity receptor for kininogen-1/HMWK .

• Functional KKS Activation Assays: Assess KKS activation through measurement of kallikrein activity and subsequent bradykinin generation to determine KRT1's regulatory effects on this pathway.

• In Vivo Validation: Correlate cellular findings with in vivo models (e.g., DSS-induced colitis) by analyzing tissue samples for KKS components and activation markers before and after KRT1 treatment .

These methodologies provide complementary insights into how KRT1 modulates the KKS, connecting molecular interactions to functional outcomes in inflammatory processes.

How do I interpret contradictory results between cell proliferation and barrier function studies using KRT1 antibodies?

Interpreting contradictory results between KRT1 antibody effects on cell proliferation and barrier function requires consideration of several biological and methodological factors. Research has demonstrated that KRT1 antibodies can simultaneously inhibit cell proliferation while also disrupting barrier function through reduction of tight junction proteins . This apparent contradiction reflects KRT1's complex role in cellular homeostasis.

When encountering such discrepancies, consider:

• Concentration-Dependent Effects: Different cellular processes may have varying sensitivity thresholds to KRT1 inhibition. For example, at 1 ng/mL, KRT1 antibody may affect certain pathways while having minimal impact on others .

• Temporal Dynamics: Proliferation inhibition may precede barrier disruption or vice versa. Time-course experiments are essential for distinguishing primary from secondary effects.

• Cell Type Specificity: KRT1 functions differently across cell types. Effects observed in Caco-2 cells may differ from other epithelial cells due to varying baseline KRT1 expression and function .

• Pathway Crosstalk: KRT1 regulates multiple signaling pathways simultaneously, including PKC/SRC via integrin beta-1 binding and the Wnt/β-catenin pathway . Differential activation of these pathways may produce seemingly contradictory outcomes.

• Antibody Specificity: Different epitope recognition by various KRT1 antibodies may selectively inhibit certain functions while preserving others.

To resolve contradictions, implement time-course studies, concentration gradients, and pathway-specific inhibitors to delineate the sequence and mechanism of events following KRT1 modulation.

What factors should be considered when translating in vitro KRT1 findings to in vivo models?

Translating in vitro findings about KRT1 to in vivo models requires careful consideration of several factors to ensure biological relevance and experimental validity. Based on successful translation strategies in KRT1 research, particularly in inflammatory models, the following factors are critical:

• Dosage Adjustment: In vitro effective concentrations (1-10 ng/mL for KRT1 antibody) require physiological scaling for in vivo administration, accounting for distribution volume, clearance rates, and tissue penetration.

• Administration Route: For intestinal barrier studies, consider both systemic (intraperitoneal) and local (oral/rectal) administration routes to target specific tissue compartments where KRT1 functions .

• Model Selection: Choose disease models that reflect the pathophysiological context where KRT1 functions. For inflammatory studies, DSS-induced colitis effectively demonstrates KRT1's protective effects on colonic injury and barrier function .

• Endpoint Harmonization: Align in vitro and in vivo endpoints by measuring comparable parameters. For barrier function, tight junction protein expression (occludin, ZO-1, claudin) should be assessed in both systems .

• Temporal Considerations: In vivo processes typically occur on different time scales than in vitro events. Design experiments with appropriate time points to capture acute versus chronic effects of KRT1 modulation.

• Microenvironment Complexity: Account for the complex in vivo environment where KRT1 interacts with multiple cell types and systems simultaneously, unlike controlled in vitro conditions.

Successful translation has been demonstrated in studies where KRT1 treatment mitigated DSS-induced colitis symptoms, increased colon length, alleviated weight loss, and suppressed inflammatory cytokines in mice, confirming in vitro observations of KRT1's protective effects .

How can KRT1 monoclonal antibodies be used to investigate epithelial-mesenchymal transition in cancer research?

KRT1 monoclonal antibodies offer valuable tools for investigating epithelial-mesenchymal transition (EMT) in cancer research through multiple experimental approaches. KRT1, as a differentiation-specific keratin predominant in stratified epithelia , represents an important epithelial marker whose expression changes during EMT.

To investigate EMT using KRT1 antibodies, researchers should consider:

• Expression Profiling: Use immunohistochemistry with KRT1 monoclonal antibodies (recommended dilution 1:20-1:200) to analyze expression patterns in normal versus cancerous tissues. During EMT, epithelial markers including keratins typically decrease while mesenchymal markers increase.

• Co-localization Studies: Implement dual immunofluorescence labeling using conjugated KRT1 antibodies together with mesenchymal markers to identify cells undergoing EMT. CF®568 or CF®594 conjugates provide excellent options for co-staining experiments with minimal spectral overlap .

• Functional Studies: Apply KRT1 antibodies at varying concentrations (1-10 ng/mL) to cancer cell lines and monitor changes in cell morphology, migration, and invasion capabilities . KRT1's interaction with integrin beta-1 and subsequent regulation of kinase activity (PKC and SRC) may directly influence EMT processes.

• Signaling Pathway Analysis: Investigate how KRT1 modulates signaling pathways involved in EMT, particularly the Wnt/β-catenin pathway , which plays a dual role in both EMT and epithelial regeneration.

• 3D Culture Systems: Apply KRT1 antibodies in three-dimensional culture systems that better recapitulate tissue architecture to assess EMT in a more physiologically relevant context.

These approaches leverage KRT1 antibodies to delineate the complex role of this keratin in maintaining epithelial identity and its alterations during cancer progression.

What emerging roles of KRT1 in immune modulation warrant further investigation?

Recent discoveries about KRT1's role in immune modulation open several promising avenues for future research. Based on current findings, the following emerging areas warrant in-depth investigation:

• KRT1-Mediated Regulation of Inflammatory Cytokines: Evidence indicates that KRT1 suppresses inflammatory cytokines such as IL-1, IL-6, and TNF-α in DSS-induced colitis models . Future research should explore the molecular mechanisms underlying this regulation and extend these investigations to other inflammatory conditions.

• Interaction with the Kallikrein Kinin System: KRT1's role in modulating the KKS, particularly its inhibition of bradykinin activation and regulation of high molecular weight kininogen , represents a novel immune regulatory mechanism. Further studies should delineate this pathway in various inflammatory contexts.

• KRT1-C1QBP Complex as Immune Regulator: The formation of a complex between KRT1 and C1QBP creates a high-affinity receptor for kininogen-1/HMWK . This interaction's implications for complement activation and innate immunity remain largely unexplored.

• Epithelial-Immune Cell Cross-talk: How KRT1 expression in epithelial cells influences resident and infiltrating immune cells requires investigation, particularly in the context of barrier tissues like the intestine and skin.

• Post-translational Modifications of KRT1: Research into how phosphorylation, glycosylation, or other modifications of KRT1 alter its immune regulatory functions could reveal novel mechanisms of immune modulation.

• Therapeutic Targeting: Development of selective KRT1 modulators (beyond antibodies) warrants exploration for treating inflammatory conditions, particularly ulcerative colitis where KRT1 has demonstrated protective effects .

These research directions promise to expand our understanding of KRT1 beyond its structural role in epithelia to its emerging functions as an immune regulator.