KREMEN1 Antibody

What are the optimal applications for KREMEN1 antibodies in experimental settings?

KREMEN1 antibodies have demonstrated efficacy in several laboratory techniques with application-specific considerations. Western Blot applications typically require dilutions between 1:1000-1:6000, with validated reactivity in human, mouse, and rat samples . For immunohistochemistry applications, KREMEN1 antibodies have been successfully used in fixed paraffin-embedded sections, as demonstrated with human colon cancer tissue, typically at concentrations around 15 μg/mL with overnight incubation at 4°C . ELISA applications also show reliable performance. When designing experiments, it's essential to validate the antibody in your specific experimental system, as the required dilution may be sample-dependent. Starting with manufacturer-recommended dilutions and optimizing through titration experiments will ensure optimal signal-to-noise ratios for your specific application .

How should KREMEN1 antibodies be stored to maintain optimal activity?

KREMEN1 antibodies require specific storage conditions to maintain their functional integrity. Most commercial preparations should be stored at -20°C, where they typically remain stable for one year after shipment . The storage buffer generally consists of PBS with 0.02% sodium azide and 50% glycerol at pH 7.3 to maintain stability . For antibodies supplied in small volumes (e.g., 20μl), preparations may contain 0.1% BSA to prevent protein loss through adsorption to storage containers . Importantly, aliquoting is generally unnecessary for -20°C storage of glycerol-containing preparations, reducing handling-related degradation risk. When working with the antibody, minimize freeze-thaw cycles by keeping working aliquots at 4°C for short-term use while maintaining the stock at -20°C.

What controls should be included when using KREMEN1 antibodies for experimental validation?

Proper experimental controls are essential for validating KREMEN1 antibody results. For positive controls, mouse and rat liver tissues have been confirmed to express detectable levels of KREMEN1 by Western blot . Human colon cancer tissue sections can serve as positive controls for immunohistochemistry applications . Negative controls should include: (1) isotype controls using non-specific IgG from the same host species as the KREMEN1 antibody; (2) secondary antibody-only controls to assess non-specific binding; and (3) when possible, KREMEN1-knockout or knockdown samples to confirm antibody specificity. For advanced validation, pre-absorption of the antibody with its immunizing antigen (KREMEN1 fusion protein) can demonstrate binding specificity. Incorporating these controls ensures the observed signals genuinely represent KREMEN1 detection rather than experimental artifacts.

How can KREMEN1 antibodies be utilized to investigate homo- and heterodimerization dynamics?

KREMEN1 undergoes complex dimerization processes that regulate its biological functions, particularly its apoptotic activity. To investigate these dynamics, researchers can employ multiple complementary approaches. Co-immunoprecipitation experiments using differentially tagged KREMEN1 constructs (such as HA-KREMEN1 and Flag-KREMEN1) have successfully demonstrated KREMEN1 homodimerization, yielding distinct bands at approximately 120 kDa (dimers) and above 200 kDa (potential trimers or complexes) following BS3 crosslinking . For heterodimerization studies, particularly with KREMEN2, competition experiments can be designed where cells are transfected with tagged KREMEN1 constructs along with potential binding partners, followed by co-immunoprecipitation to assess displacement of KREMEN1 homodimers . To visualize these interactions in cellular contexts, immunofluorescence colocalization studies using KREMEN1 antibodies combined with antibodies against potential binding partners provide spatial information about interaction sites. These approaches collectively allow researchers to dissect the molecular mechanisms controlling KREMEN1's diverse biological functions.

What methodological approaches are recommended for investigating KREMEN1-mediated apoptotic signaling?

KREMEN1's function as a dependence receptor that triggers cell death in the absence of its ligand Dickkopf1 (Dkk1) requires specific methodological approaches for thorough investigation. Caspase-3 immunostaining serves as a primary readout for KREMEN1-induced apoptosis in transfected cells . This approach can be complemented with TUNEL assays or Annexin V/PI staining for comprehensive apoptosis assessment. To investigate the structural determinants of KREMEN1's apoptotic function, truncation constructs (separating extracellular, transmembrane, and intracellular domains) can be generated and tested for their ability to induce apoptosis compared to full-length KREMEN1 . The regulatory role of ligand binding can be evaluated by supplementing experimental systems with recombinant Dkk1, which should inhibit KREMEN1-induced cell death. For studying competitive inhibition by KREMEN2, co-transfection experiments can be designed with varying ratios of KREMEN1 and KREMEN2, followed by assessment of apoptotic markers . Flow cytometry provides a quantitative approach for analyzing apoptosis across cell populations under these various experimental conditions.

How can researchers investigate the interaction between KREMEN1 and viral proteins in receptor studies?

Recent findings identifying KREMEN1 as an alternative receptor for SARS-CoV-2 and certain enteroviruses highlight the importance of methodological approaches for viral-receptor interaction studies. S-pseudotyped viral particles provide a practical system for investigating KREMEN1-mediated viral entry without requiring high-containment facilities . When combined with KREMEN1 overexpression or knockout systems (such as CRISPR-Cas9 edited cell lines), this approach allows quantitative assessment of KREMEN1's contribution to viral entry. Co-localization studies using confocal microscopy with KREMEN1 antibodies and viral protein antibodies (such as SARS-CoV-2 Spike) can visualize direct interactions between virus and receptor . For more detailed binding kinetics, surface plasmon resonance or biolayer interferometry using purified KREMEN1 extracellular domain and viral proteins allows precise measurement of binding affinities and association/dissociation rates. To validate findings in physiologically relevant systems, lentiviral transduction of human KREMEN1 into animal models followed by viral challenge provides in vivo confirmation of receptor function . These complementary approaches collectively enable thorough characterization of KREMEN1's role in viral entry mechanisms.

What methodologies are recommended for investigating KREMEN1's tumor suppressor function?

KREMEN1 demonstrates tumor suppressor activity by preventing cancer cell survival in ligand-poor environments through its apoptotic function . To investigate this role, multiple experimental approaches can be employed. Analysis of KREMEN1 expression levels across tumor vs. normal tissue samples provides initial insights, with evidence showing decreased expression in various cancers . This can be accomplished through immunohistochemistry with KREMEN1 antibodies on tissue microarrays or Western blot analysis of tumor lysates compared to matched normal tissue. For functional studies, gain-of-function experiments involving KREMEN1 overexpression in cancer cell lines followed by assessment of proliferation, colony formation, and apoptotic markers helps establish causative relationships. Conversely, KREMEN1 knockdown or knockout in normal cells can reveal whether its loss promotes oncogenic phenotypes. To investigate mechanisms linking KREMEN1 to apoptotic resistance, researchers should examine the correlation between KREMEN1 expression and its binding partners, particularly Dkk1 and KREMEN2, which have been shown to counteract KREMEN1's apoptotic function . In vivo xenograft studies with KREMEN1-modulated cancer cells provide the most comprehensive assessment of its tumor suppressor function.

How can KREMEN1 antibodies be used to investigate its role in viral pathogenesis?

KREMEN1 has been identified as an alternative receptor for SARS-CoV-2 and certain enteroviruses, providing a crucial mechanism for ACE2-independent viral entry . For investigating KREMEN1's role in viral pathogenesis, immunohistochemistry with KREMEN1 antibodies on tissue sections from infection models or patient samples helps establish correlation between KREMEN1 expression patterns and viral tropism. Flow cytometry with KREMEN1 antibodies can quantify receptor expression levels across different cell populations to identify susceptible cell types. Neutralization experiments using KREMEN1 antibodies alone or in combination with antibodies targeting other receptors (creating "cocktails") assess the relative contribution of different entry pathways and may identify potential therapeutic approaches . For molecular interaction studies, site-directed mutagenesis of KREMEN1 followed by viral binding and entry assays can identify critical residues for virus-receptor interactions, such as the D90 residue that engages with the conserved K140 residue in KRM1-dependent enteroviruses . In organoid systems, which better reflect the complexity of natural tissues, KREMEN1 antibody blockade experiments provide physiologically relevant insights into entry mechanisms .

What experimental approaches can determine KREMEN1's role in hand-foot-and-mouth disease and ectodermal dysplasia?

KREMEN1 variations and mutations have been associated with ectodermal dysplasia and hand-foot-and-mouth disease (HFMD) . For investigating these connections, genetic analysis should be combined with functional studies using KREMEN1 antibodies. In HFMD research, immunohistochemistry with KREMEN1 antibodies on skin lesion biopsies can correlate receptor expression with sites of enterovirus infection. The crucial role of the conserved VP2 residue K140 in KREMEN1-dependent enteroviruses can be studied using viral mutants in both in vitro and in vivo infection models . For ectodermal dysplasia studies, researchers should analyze KREMEN1 expression patterns during embryonic development using antibodies on developmental tissue series. Patient-derived cells carrying KREMEN1 mutations can be analyzed for altered receptor expression, localization, and function compared to wild-type controls. Mechanistically, KREMEN1's involvement in Wnt signaling during organ development (including nervous system, limbs, and liver) suggests examining dysregulated Wnt pathway components in affected tissues . For each condition, creating animal models with equivalent KREMEN1 mutations found in human patients provides systems for studying disease progression and potential therapeutic interventions.

What are common pitfalls in Western blot detection of KREMEN1 and how can they be addressed?

Western blot detection of KREMEN1 presents several technical challenges that require specific optimization strategies. The observed molecular weight of KREMEN1 (approximately 50 kDa) may differ from its calculated molecular weight (52 kDa) due to post-translational modifications . Additionally, detection of KREMEN1 dimers and higher-order structures requires specific sample preparation approaches. For optimal detection:

• Sample preparation should include proper cell lysis in the presence of protease inhibitors to prevent degradation of KREMEN1.

• For detecting homo- and heterodimers, chemical crosslinking with BS3 or similar reagents prior to lysis helps stabilize these typically transient interactions .

• The appropriate antibody dilution range for Western blot applications is typically between 1:1000-1:6000, requiring optimization for specific sample types .

• KREMEN1's transmembrane nature may necessitate longer transfer times or specialized transfer conditions for efficient movement to membranes.

• Background issues can be minimized by extending blocking times and using 5% non-fat milk or BSA in TBS-T.

• Validated positive controls include mouse and rat liver tissue lysates, which reliably express detectable KREMEN1 levels .

When troubleshooting weak or absent signals, consider employing protein enrichment methods such as immunoprecipitation before Western blot analysis, particularly for samples with naturally low KREMEN1 expression.

How should researchers address cross-reactivity concerns with KREMEN1 antibodies?

Cross-reactivity is a significant concern when working with KREMEN1 antibodies, particularly given its structural similarity to KREMEN2 and the potential for non-specific binding. To address these issues:

• Validate antibody specificity using KREMEN1 knockout or knockdown samples compared to wild-type controls.

• When studying KREMEN1 in systems that may express KREMEN2, verify antibody specificity by testing against recombinant KREMEN2 protein or KREMEN2-overexpressing cells.

• Perform pre-absorption controls by incubating the antibody with purified KREMEN1 antigen prior to application – this should eliminate specific binding signals.

• For immunohistochemistry applications, include isotype controls and secondary antibody-only controls to identify non-specific binding.

• When possible, confirm results using antibodies raised against different epitopes of KREMEN1 to reduce epitope-specific artifacts.

• For commercially available antibodies, select those validated against the specific application and species of interest, such as those confirmed to react with human, mouse, and rat samples .

These validation steps are particularly important when investigating KREMEN1 in novel tissue types or experimental conditions where expression patterns have not been previously characterized.

What is the recommended methodology for detecting KREMEN1 in fixed tissues?

Detecting KREMEN1 in fixed tissue samples requires specific methodological considerations to ensure sensitive and specific staining. Based on validated protocols:

• Paraffin-embedded tissue sections fixed with formalin provide reliable results for KREMEN1 immunohistochemistry .

• Antigen retrieval steps are critical – heat-induced epitope retrieval using citrate buffer (pH 6.0) is typically effective for KREMEN1 detection.

• Primary antibody concentrations around 15 μg/mL with overnight incubation at 4°C have been validated for human tissues .

• Detection systems such as HRP-DAB provide good visualization of KREMEN1 expression patterns, with hematoxylin counterstaining to visualize tissue architecture .

• For double immunofluorescence studies investigating KREMEN1 interaction with binding partners, sequential staining protocols may be necessary to avoid cross-reactivity between detection systems.

• Positive control tissues should include samples known to express KREMEN1, such as human colon cancer tissue .

• When quantifying KREMEN1 expression in tissues, standardized scoring systems considering both staining intensity and percentage of positive cells provide more reliable results than subjective assessments.

These approaches enable reliable detection of KREMEN1 in fixed tissues for diagnostic, prognostic, or basic research applications.

How do KREMEN1, ASGR1 and ACE2 receptors cooperatively facilitate SARS-CoV-2 entry?

Recent groundbreaking research has identified KREMEN1 as part of an alternative entry pathway for SARS-CoV-2, functioning alongside ASGR1 and the canonical ACE2 receptor. This discovery has significant implications for understanding viral tropism and developing therapeutic strategies. Experimentally, ectopic expression of KREMEN1 in ACE2-knockout cells demonstrates its sufficiency for SARS-CoV-2 entry, though this pathway appears specific to SARS-CoV-2 and not SARS-CoV . KREMEN1 facilitates efficient attachment of S-pseudotyped SARS-CoV-2 particles to cell surfaces, with co-localization studies confirming direct interaction between S protein and KREMEN1 .

The collective expression of ACE2, SARS-CoV-2 Glycoprotein Receptor 1 (ASGR1), and KREMEN1 (termed the "ASK receptome") correlates more strongly with SARS-CoV-2 susceptibility than any individual receptor at both cellular and tissue levels . This suggests SARS-CoV-2 utilizes distinct receptor combinations to enter different cell types, potentially explaining its broad tissue tropism. Importantly, neutralizing antibody cocktails targeting all three receptors provide more substantial blockage of infection in human lung organoids compared to individual antibodies . This finding offers valuable direction for therapeutic development, suggesting that comprehensive receptor blockade strategies may provide more effective protection against infection than approaches targeting individual receptors.

What is the significance of the conserved VP2 residue K140 in KREMEN1-dependent enterovirus infections?

The VP2 residue K140 has emerged as a critical determinant in KREMEN1-dependent enterovirus infections. This residue is completely conserved among all KREMEN1-dependent enteroviruses, including coxsackievirus A2-A6 and A12, highlighting its functional importance . Mutational analysis has confirmed that K140 is essential for viral infection by these enterovirus strains. The mechanistic basis for this requirement involves direct interaction between K140 and the D90 residue of KREMEN1, which plays a crucial role in mediating enterovirus infections .

This discovery has expanded our understanding of viral receptor usage, with experimental work demonstrating that coxsackievirus A8, which also possesses the conserved K140 residue, utilizes KREMEN1 as its receptor . This finding provides a molecular basis for predicting receptor usage among enteroviruses based on conservation of key residues. From a translational perspective, the identification of this absolutely conserved viral residue presents a potential target for broad-spectrum antiviral therapies against hand-foot-and-mouth disease (HFMD)-causing enteroviruses. Targeting the K140-D90 interaction interface could theoretically inhibit multiple KREMEN1-dependent enteroviruses simultaneously, offering a strategy for developing pan-enterovirus interventions with broader coverage than approaches targeting strain-specific epitopes.

How is KREMEN1-induced cell death regulated by homo- and heterodimerization?

KREMEN1 functions as a dependence receptor that can trigger apoptosis in the absence of its ligand Dickkopf1 (Dkk1), with this pro-apoptotic activity regulated through complex dimerization mechanisms. Experimental evidence demonstrates that KREMEN1 forms homodimers through its extracellular domain, with crosslinking studies revealing distinct bands at approximately 120 kDa (dimers) and above 200 kDa (potential trimers or complexes with partners) . These homodimers are crucial for KREMEN1's apoptotic signaling, as mutations disrupting dimerization also abolish cell death induction.

The apoptotic activity of KREMEN1 is negatively regulated through two primary mechanisms. First, binding of its ligand Dkk1 disrupts KREMEN1 homodimers, preventing apoptotic signaling . Second, heterodimerization with KREMEN2 strongly reduces KREMEN1 homodimerization and efficiently silences Caspase-3 activation, with the extracellular domain of KREMEN2 being necessary and sufficient for this inhibitory effect . This regulatory mechanism appears relevant in cancer biology, where KREMEN1 expression is frequently decreased while KREMEN2 expression is increased across multiple cancer types, potentially preventing KREMEN1-induced apoptosis in tumor cells . Differential gene expression analyses show KREMEN2 upregulation in >80% of tumor samples compared to normal tissue across various cancer types, with particularly dramatic increases (>10-fold) in lung squamous cell carcinoma . These findings suggest targeting KREMEN2 to restore KREMEN1-mediated apoptosis could represent a novel therapeutic strategy for certain cancers.