wscd2 Antibody

What is known about the WSCD2 protein structure and domains?

WSCD2 is a 565 amino acid single-pass membrane protein containing two WSC domains. The WSC domain, named after yeast WSC1-WSC4 (cell wall integrity and stress response components 1-4), is a putative carbohydrate binding domain spanning approximately 90 amino acids. Each WSC domain contains eight conserved cysteine residues predicted to form disulfide bridges. These domains are often found alongside other domains including LDL-receptor class A, PKD, and C-type lectin domains. The predicted molecular weight of WSCD2 is approximately 64 kDa, and it is classified as a transmembrane protein .

Which antibody formats are available for WSCD2 detection?

Researchers have multiple antibody options for WSCD2 detection, including:

• Rabbit polyclonal antibodies: Generated using synthetic peptides directed toward the C-terminal region of human WSCD2, with reactivity primarily against human targets .

• Mouse monoclonal antibodies (e.g., clone 9-F3): Developed using recombinant protein with Human WSCD2 amino acids 50-353/565, exhibiting broader species reactivity including human, mouse, and rat .

The choice between polyclonal and monoclonal depends on the specific experimental requirements - polyclonal antibodies offer broader epitope recognition, while monoclonal antibodies provide higher specificity for a single epitope .

What applications have been validated for WSCD2 antibodies?

Current WSCD2 antibodies have been validated for several standard applications:

When performing Western blotting, researchers should anticipate detecting a band at approximately 64 kDa, which corresponds to the predicted protein size of WSCD2. Validation data has confirmed detection in A549 cell line lysates, making this a reliable positive control for experimental setup .

How should researchers optimize antibody concentration for WSCD2 detection in Western blotting?

For Western blotting applications, optimization is critical since WSCD2's function remains largely uncharacterized. Begin with manufacturer-recommended dilutions, typically using purified antibody supplied in 1x PBS buffer with 0.09% (w/v) sodium azide and 2% sucrose. For rabbit polyclonal antibodies, start with dilutions in the range specified by the manufacturer. For mouse monoclonal antibodies like clone 9-F3 (concentration typically 2 mg/mL), perform a dilution series (1:500, 1:1000, 1:2000) to determine optimal signal-to-noise ratio.

When optimizing, include positive controls such as A549 cell lysates where WSCD2 has been successfully detected. Additionally, due to the transmembrane nature of WSCD2, ensure complete protein denaturation and consider using specialized buffers for membrane proteins to maximize epitope exposure. Monitor for non-specific binding, particularly with polyclonal antibodies, and adjust blocking conditions accordingly .

What considerations are important for cross-species reactivity in WSCD2 antibody selection?

When designing experiments involving multiple species, researchers should carefully evaluate the immunogen sequence homology provided by manufacturers. Available data shows varying degrees of conservation across species:

For studies involving mice, note the 85% homology which may result in reduced antibody affinity. When working with zebrafish models (77% homology), additional validation experiments are strongly recommended. The mouse monoclonal antibody (clone 9-F3) has been specifically validated for reactivity with human, mouse, and rat, making it suitable for comparative studies across these species. Researchers should perform species-specific validation using appropriate positive controls regardless of reported reactivity .

How can researchers assess WSCD2 antibody specificity?

Due to the unknown function of WSCD2, establishing antibody specificity is particularly critical. Implement a multi-faceted approach:

• Peptide competition assays: Pre-incubate the antibody with the immunizing peptide (for polyclonal antibodies generated against synthetic peptides directed towards the C-terminal of human WSCD2).

• Knockdown/knockout validation: Use siRNA knockdown or CRISPR/Cas9 knockout models to confirm signal disappearance.

• Orthogonal detection methods: Compare results from antibodies targeting different epitopes of WSCD2.

• Mass spectrometry validation: Perform immunoprecipitation followed by mass spectrometry to confirm the identity of the detected protein.

• Cross-reference with tagged overexpression systems: Compare detection patterns between endogenous WSCD2 and epitope-tagged overexpression constructs.

These approaches are particularly important given that the function of WSCD2 remains unknown, and validation data in the literature is limited .

How should researchers address conflicting WSCD2 antibody results?

When confronted with conflicting results, implement a systematic troubleshooting approach:

• Antibody verification: Re-validate antibody specificity using peptide competition or knockout controls.

• Epitope accessibility: Consider that the C-terminal epitopes used for some antibodies (amino acids 536-565) may have differential accessibility depending on experimental conditions or protein modifications.

• Isoform specificity: Note that two isoforms of WSCD2 exist as a result of alternative splicing events, which could explain differential detection patterns.

• Cell/tissue-specific expression: Validate expression in your specific model system, as expression levels may vary significantly across different cell types.

• Technical parameters: Evaluate fixation methods (for IF/IHC) or lysis conditions (for WB) that might affect epitope recognition.

Document all experimental conditions thoroughly to facilitate troubleshooting and consult published literature on epitope repertoire studies that have included WSCD2 to contextualize your findings .

What data analysis considerations are important when studying WSCD2 in large-scale proteomic studies?

In large-scale proteomic or antibody repertoire studies incorporating WSCD2:

• Control for epitope bias: When using phage display systems like those described in antibody repertoire studies, be aware that the system may introduce biases based on peptide presentation.

• Population variance: Consider population-wide antibody diversity against WSCD2, as suggested by large-scale studies of serum antibody epitope repertoires.

• Statistical thresholds: Apply appropriate statistical corrections (e.g., Bonferroni correction) when analyzing large datasets, particularly in genome-wide studies where WSCD2 variants have been implicated.

• Functional validation: Due to the unknown function of WSCD2, correlate proteomic findings with functional assays to establish biological relevance.

• Longitudinal analysis: When possible, incorporate longitudinal data (e.g., t=0 and t=5 years) as mentioned in antibody repertoire studies to assess stability of findings over time.

How can researchers interpret WSCD2 antibody signals in the context of its unknown function?

When interpreting WSCD2 antibody signals without established functional context:

• Correlative analysis: Assess co-expression or co-localization with proteins of known function, particularly those containing similar WSC domains.

• Subcellular localization: Use the single-pass transmembrane nature of WSCD2 as a starting point to confirm appropriate subcellular localization using imaging techniques.

• Domain-based inference: Leverage knowledge of the WSC domain as a putative carbohydrate binding domain to design functional assays testing this potential activity.

• Genetic association context: Consider the suggestive overlapping signals between linkage and association observed at rs59849892 in WSCD2 in relation to cardiometabolic phenotypes.

• Cross-reference with public databases: Compare experimental findings with information in resources like the Immune Epitope Database (IEDB) mentioned in the antibody repertoire studies.

This interpretive framework provides a foundation for hypothesis generation despite the limited understanding of WSCD2's biological role .

What is the potential significance of WSCD2 in cardiometabolic research?

Genome-wide linkage and association analyses have identified suggestive overlapping signals at rs59849892 in the WSCD2 gene related to cardiometabolic phenotypes. Researchers investigating this connection should consider:

• Experimental design incorporating metabolic parameters: Design studies that assess WSCD2 expression or localization in response to metabolic challenges.

• Tissue-specific expression analysis: Determine expression patterns in metabolically relevant tissues (adipose, liver, muscle, pancreas).

• Potential functional pathways: Explore connections to insulin signaling, cholesterol metabolism, or other cardiometabolic pathways.

• Model systems: Consider using diet-induced obesity models or diabetic models to assess regulation of WSCD2.

• Clinical correlation: Design translational studies that correlate WSCD2 expression or polymorphisms with clinical cardiometabolic parameters.

These approaches may help elucidate the potential role of WSCD2 in cardiometabolic conditions and provide context for the genetic associations observed in population studies .

How can researchers leverage antibody epitope repertoire studies to advance WSCD2 research?

Large-scale antibody epitope repertoire studies that have included WSCD2 offer novel research opportunities:

• Epitope mapping: Use information from these studies to identify immunodominant regions of WSCD2 that may indicate functionally or structurally important domains.

• Population immunity analysis: Explore whether natural antibody responses against WSCD2 correlate with health status or disease predisposition.

• Cross-reactivity investigation: Determine whether antibodies against WSCD2 cross-react with microbial proteins, suggesting potential molecular mimicry mechanisms.

• Longitudinal stability: Assess the stability of anti-WSCD2 antibody responses over time as a potential biomarker for specific conditions.

• Integration with microbiome data: Correlate anti-WSCD2 antibody responses with microbiome composition, particularly since WSC domains are associated with cell wall components.

These approaches leverage population-scale antibody repertoire data to generate novel hypotheses about WSCD2 function and potential relevance in human health and disease .

What are the critical gaps in current WSCD2 research?

Several critical knowledge gaps exist in WSCD2 research:

• Functional characterization: The fundamental biological function of WSCD2 remains unknown, presenting a significant obstacle to targeted research.

• Structure-function relationships: The specific roles of the two WSC domains in WSCD2 and their interaction partners have not been characterized.

• Tissue-specific expression patterns: Comprehensive analysis of expression across different tissues and cell types is lacking.

• Regulatory mechanisms: Factors controlling WSCD2 expression and potential post-translational modifications remain unexplored.

• Disease associations: Despite genetic associations with cardiometabolic traits, the mechanistic relationships between WSCD2 and disease states are not established.

Addressing these gaps requires interdisciplinary approaches combining structural biology, functional genomics, and translational research with appropriate antibody-based detection methods .

What methodological advances would enhance WSCD2 antibody-based research?

Future methodological developments could significantly advance WSCD2 research:

• Development of isoform-specific antibodies: Creating tools that distinguish between the two known splice variants would enable more precise analysis.

• Domain-specific antibodies: Generating antibodies that specifically recognize each WSC domain would facilitate structural and functional studies.

• Proximity labeling approaches: Adapting antibodies for BioID or APEX2 proximity labeling would help identify interaction partners.

• Live-cell imaging compatible formats: Developing non-disruptive labeling approaches for tracking WSCD2 in living cells.

• Phospho-specific antibodies: Creating tools that detect potential regulatory phosphorylation sites as they are identified.