SCEL Antibody

What is SCEL and what is its biological function?

SCEL (sciellin) is a precursor to the cornified envelope of terminally differentiated keratinocytes. The protein localizes to the periphery of cells and is believed to function in the assembly or regulation of proteins in the cornified envelope. The LIM domain within SCEL may be involved in homotypic or heterotypic associations and may function to localize sciellin to the cornified envelope. SCEL is highly expressed in esophagus and is also found in keratinocytes, amniotic tissue, foreskin stratum spinosum and stratum granulosum, hair follicle, and nail tissue . The protein can become cross-linked to membrane proteins by transglutaminase, suggesting its role in maintaining epithelial structural integrity .

What are the molecular characteristics of human SCEL protein?

Human SCEL has a canonical protein length of 688 amino acid residues with a calculated molecular weight of 75-77.6 kDa, though the observed molecular weight in experimental conditions typically ranges from 70-78 kDa . The protein contains functional domains that contribute to its structural role in epithelial tissues. Up to three different isoforms have been reported for this protein, suggesting alternative splicing may contribute to tissue-specific functions . The gene is identified with NCBI Gene ID 8796, and the protein has UniProt ID O95171 .

What types of SCEL antibodies are available for research applications?

SCEL antibodies are available in both polyclonal and monoclonal formats, with various host species including rabbit and mouse. These antibodies target different epitopes within the SCEL protein, such as the N-terminal (AA 2-97), central region (AA 260-289), and C-terminal (AA 581-688) domains . The diversity of available antibodies allows researchers to select reagents optimized for specific applications or targeting particular protein domains. Most commonly, these antibodies are unconjugated, though some biotinylated versions are available for specialized detection methods .

What are the recommended applications for SCEL antibodies?

SCEL antibodies are validated for multiple applications including Western Blot (WB), Immunohistochemistry (IHC), Immunofluorescence (IF/ICC), Flow Cytometry (FC), and ELISA . For Western blotting, recommended dilutions typically range from 1:1000 to 1:5000. For immunohistochemistry applications, dilutions of 1:50 to 1:500 are commonly suggested. Immunofluorescence and immunocytochemistry typically require dilutions between 1:50 and 1:500. For flow cytometry applications, a typical concentration would be 0.25 μg per 10^6 cells in a 100 μl suspension . These recommendations provide starting points, but optimization for specific experimental conditions is recommended.

What tissue and cell types show positive reactivity with SCEL antibodies?

Based on validation studies, SCEL antibodies show positive Western blot detection in human saliva tissue, A431 cells, human skin tissue, and mouse skin tissue . Positive immunohistochemistry reactivity has been documented in human renal cell carcinoma tissue. Immunofluorescence and immunocytochemistry applications have demonstrated positive results in HepG2 cells, while flow cytometry has been validated using HeLa cells . The SCEL marker can also be used to identify Alveolar Type I Cells, expanding its utility in respiratory research .

How should SCEL antibodies be stored for optimal stability?

SCEL antibodies should typically be stored at -20°C for long-term preservation. The antibodies remain stable for approximately one year after shipment when properly stored. For short-term storage up to two weeks, refrigeration at 2-8°C is recommended . The antibodies are commonly supplied in PBS buffer containing 0.02-0.09% sodium azide and 50% glycerol at pH 7.3, which helps maintain stability . For the 20μl size antibody preparations, some formulations contain 0.1% BSA. Aliquoting is generally unnecessary for -20°C storage, though it may be beneficial for antibodies that will undergo multiple freeze-thaw cycles .

How can cross-reactivity between SCEL and related proteins be assessed and controlled?

Cross-reactivity assessment for SCEL antibodies requires comprehensive validation using positive and negative control tissues with known SCEL expression profiles. To control for potential cross-reactivity, researchers should conduct preliminary testing using knockout or knockdown models where SCEL expression is absent or significantly reduced. Pre-adsorption experiments, where the antibody is pre-incubated with excess purified SCEL protein before use in the intended application, can help identify non-specific binding. Conducting parallel experiments with multiple SCEL antibodies targeting different epitopes can provide confirmatory evidence of specificity, particularly when consistent results are observed across different antibody clones . Western blot analysis should reveal bands at the expected molecular weight (70-78 kDa), and any additional bands should be carefully investigated for potential splice variants or protein modifications.

What are the considerations for using SCEL antibodies in multi-color fluorescence microscopy?

When designing multi-color fluorescence microscopy experiments with SCEL antibodies, spectral overlap must be carefully considered. Since SCEL is primarily localized to the cytoplasm and membrane, co-staining with markers from these compartments requires proper controls to distinguish genuine colocalization from coincidental spatial proximity. For optimal results in immunofluorescence applications, a dilution range of 1:50-1:500 is recommended as a starting point . When analyzing SCEL in stratified epithelia, Z-stack imaging may be necessary to properly visualize the differential expression across layers. Autofluorescence from keratin-rich tissues can interfere with signal detection, so appropriate background subtraction and autofluorescence quenching techniques should be employed. For co-localization studies with other cornified envelope proteins, careful selection of compatible secondary antibodies and sequential staining protocols may be required to prevent cross-reactivity.

How can SCEL antibodies be employed in the study of cornified envelope assembly during epithelial differentiation?

To study cornified envelope assembly using SCEL antibodies, researchers should implement time-course experiments tracking SCEL localization during keratinocyte differentiation. This requires establishing in vitro differentiation systems using calcium switch methods or 3D organotypic culture models that recapitulate stratified epithelial architecture. Immunoprecipitation experiments using SCEL antibodies (particularly those validated for IP such as antibodies targeting AA 519-685) can identify protein-protein interaction partners during envelope formation . Correlative microscopy combining immunofluorescence with electron microscopy can provide insights into the ultrastructural organization of SCEL during cornification. For functional studies, SCEL antibody-mediated inhibition experiments can be designed to assess whether blocking SCEL affects envelope formation. Comparing SCEL dynamics across different epithelial tissues (esophagus, skin, oral mucosa) using the same antibody can reveal tissue-specific aspects of cornified envelope assembly.

What antigen retrieval methods are optimal for SCEL detection in paraffin-embedded tissues?

For optimal SCEL detection in paraffin-embedded tissues, heat-induced epitope retrieval (HIER) using TE buffer at pH 9.0 is the primary recommended method . This alkaline pH environment effectively breaks protein cross-links formed during fixation, exposing epitopes for antibody binding. As an alternative, antigen retrieval may be performed with citrate buffer at pH 6.0, which may be preferable for certain tissue types or specific experimental questions . The choice between these methods should be empirically determined based on signal intensity, background levels, and tissue morphology preservation. Extended retrieval times may be necessary for heavily fixed tissues, but care should be taken to avoid over-retrieval which can compromise tissue integrity. Post-retrieval blocking steps should include both protein blocking (using BSA or serum) and peroxidase blocking when using HRP-conjugated detection systems.

What controls should be included when using SCEL antibodies in experimental protocols?

A comprehensive control strategy for SCEL antibody experiments should include multiple elements. Positive tissue controls with known SCEL expression (esophagus, skin) should be processed alongside experimental samples . Negative controls should include tissues with minimal or no SCEL expression. Antibody-specific controls should include primary antibody omission, isotype controls (rabbit IgG for rabbit host antibodies), and when available, pre-immune serum controls. For validation of antibody specificity, peptide competition assays using the immunizing peptide can confirm binding specificity. Western blot analysis should be performed to confirm the antibody detects proteins of the expected molecular weight (70-78 kDa) . For quantitative applications, standard curves using recombinant SCEL protein should be established. When interpreting immunohistochemistry results, it's important to consider that suggested dilutions (1:50-1:500) may require optimization for specific tissues and fixation protocols .

How should sample preparation be optimized for Western blot analysis of SCEL?

For optimal Western blot detection of SCEL, tissue or cell lysate preparation requires careful consideration. SCEL forms cross-links with other proteins in the cornified envelope, so lysis buffers containing sufficient detergents (such as 1% SDS) and reducing agents (like DTT or β-mercaptoethanol) are essential for complete solubilization. Protease inhibitor cocktails should be included to prevent degradation. Since SCEL has an observed molecular weight of 70-78 kDa, polyacrylamide gels with 8-10% acrylamide concentration provide optimal resolution . Extended transfer times may be necessary due to the protein's size. For blotting, recommended antibody dilutions range from 1:1000 to 1:5000, but this should be optimized for each specific antibody and experimental condition . Since SCEL may show post-translational modifications or exist in different isoforms, multiple bands may be observed, necessitating careful interpretation.

What strategies can address weak or absent signal when using SCEL antibodies?

When encountering weak or absent signals with SCEL antibodies, several optimization strategies can be implemented. First, antigen retrieval conditions should be adjusted, comparing TE buffer (pH 9.0) with citrate buffer (pH 6.0) and varying retrieval times . Antibody concentration should be systematically increased, starting from the recommended dilution (e.g., 1:500 for IHC) and testing higher concentrations if necessary . Detection system amplification using polymer-based or tyramide signal amplification can enhance sensitivity. For tissues with high endogenous biotin, avidin-biotin detection systems should be avoided or properly blocked. Incubation times and temperatures can be modified, with overnight primary antibody incubation at 4°C often yielding stronger signals than shorter incubations at room temperature. If these approaches fail, alternative SCEL antibodies targeting different epitopes should be tested, as epitope accessibility may vary across tissue preparation methods.

How can non-specific background be reduced in SCEL antibody-based detection methods?

To reduce non-specific background in SCEL antibody applications, blocking protocols should be optimized using BSA, normal serum from the secondary antibody host species, or commercial blocking reagents. For flow cytometry applications, Fc receptor blocking is critical when using the recommended concentration of 0.25 μg per 10^6 cells . When working with skin or highly keratinized tissues, additional blocking steps may be necessary to prevent non-specific binding to keratin proteins. Washing steps should be extended and performed with gentle agitation to remove unbound antibody effectively. Antibody dilution should be carefully optimized, as too concentrated antibody solutions often increase background. For immunofluorescence applications, confocal microscopy with appropriate pinhole settings can help reduce out-of-focus fluorescence. When persistent background occurs, alternative antibody clones should be considered, as some antibodies may have inherently lower background in specific applications.

What approaches can verify SCEL antibody specificity in research applications?

To verify SCEL antibody specificity, a multi-faceted validation approach is recommended. Peptide competition assays using the immunizing peptide (for example, the KLH conjugated synthetic peptide between amino acids 260-289 for certain antibodies) can confirm binding specificity . Western blot analysis should demonstrate bands at the expected molecular weight (70-78 kDa) . Parallel testing with multiple antibodies targeting different SCEL epitopes should yield consistent localization patterns. RNA interference experiments to knockdown SCEL expression should result in corresponding reduction in antibody signal. When available, SCEL knockout models provide the most definitive negative controls. Immunoprecipitation followed by mass spectrometry can independently verify that the antibody is capturing the intended target. Cross-species reactivity testing can assess conservation of the epitope, with most SCEL antibodies showing reactivity with human and mouse samples, though specific reactivity profiles vary by antibody clone .

What are the emerging applications for SCEL antibodies in current research?

SCEL antibodies are increasingly being utilized in advanced research contexts beyond traditional protein detection. Recent developments include their application in single-cell analysis to identify specific epithelial cell populations, particularly Alveolar Type I Cells in respiratory research . Their use in high-throughput screening approaches can identify compounds that modulate cornified envelope formation. In clinical research, SCEL expression analysis using validated antibodies may serve as biomarkers for epithelial differentiation in disease states. The integration of SCEL antibodies in multi-omics approaches, combining immunolabeling with proteomics or transcriptomics, provides comprehensive insights into epithelial biology. As three-dimensional tissue models become more sophisticated, SCEL antibodies are valuable tools for validating proper differentiation and stratification in engineered tissues, particularly those mimicking esophageal or epidermal structures where SCEL is highly expressed .