Recombinant Mouse DCC

Using Recombinant Mouse DCC in research applications is valuable for studying axon guidance, neuronal development, apoptosis, and cell signaling, particularly in the context of neurobiology and cancer research.

Key scientific reasons to use Recombinant Mouse DCC:

• Axon Guidance and Neural Development: DCC (Deleted in Colorectal Cancer) is a well-characterized receptor for netrins, which are critical for directing axonal growth and neuronal migration during development. Recombinant DCC allows for precise in vitro and in vivo studies of these processes, enabling mechanistic dissection of axon attraction and guidance pathways.

• Apoptosis and Dependence Receptor Function: DCC acts as a dependence receptor, inducing apoptosis in the absence of its ligand (netrin-1). Recombinant DCC can be used to investigate the molecular mechanisms of apoptosis, cell survival, and the balance between cell death and differentiation, which is relevant for both developmental biology and cancer research.

• Tumor Suppressor Studies: DCC functions as a tumor suppressor, and its loss or mutation is implicated in various cancers. Recombinant DCC enables functional assays to study its role in tumorigenesis, cell signaling, and interactions with other proteins such as UNC5 and Draxin.

• Cell Signaling Pathways: DCC mediates complex signaling cascades upon ligand binding, influencing cytoskeletal dynamics and cellular responses. Recombinant protein provides a controlled tool for biochemical and cell-based assays to map these pathways.

• Experimental Controls and Blocking Studies: Recombinant DCC can serve as a control or blocking reagent in experiments, helping to validate antibody specificity, ligand-receptor interactions, and downstream effects.

Typical applications include:

• In vitro binding assays to study netrin-DCC interactions.
• Cell culture experiments to assess DCC-mediated apoptosis or neurite outgrowth.
• Functional studies in genetically engineered mouse models to dissect DCC’s role in development or disease.
• Screening for therapeutic agents targeting DCC-related pathways.

Best practices:
Use recombinant DCC at defined concentrations for reproducibility, include appropriate controls (e.g., ligand presence/absence), and validate results with complementary genetic or pharmacological approaches.

In summary, Recombinant Mouse DCC is a critical tool for dissecting the molecular mechanisms of neural development, apoptosis, and cancer biology, providing specificity and reproducibility in experimental systems.

Yes, recombinant mouse DCC can be used as a standard for quantification and calibration in ELISA assays, provided you follow appropriate guidelines for standard preparation and validation.

Suitability as an ELISA Standard

Recombinant proteins are well-suited for ELISA standardization. The recombinant mouse DCC protein, particularly when available in Fc chimera format, can serve as a calibration standard for sandwich ELISA applications targeting the DCC antigen. This is especially valuable because recombinant proteins offer batch-to-batch consistency and reliable supply security compared to native protein sources.

Standard Preparation Considerations

When using recombinant mouse DCC as your ELISA standard, consider the following:

Formulation Selection: Recombinant proteins are available in different formulations. For ELISA standardization purposes, the protein with carrier proteins such as BSA is generally recommended for optimal performance. However, if you require carrier-free formulations for specific applications, those are also available.

Concentration Measurement: If you prepare your own standard from semi-purified recombinant protein, measure the concentration using high-performance liquid chromatography (HPLC) or other validated quantification methods to ensure accuracy.

Standard Curve Range: Establish your standard curve based on your assay requirements. Typical ELISA standard curves range from 0 to 1000 pg/mL, though this can extend to 3000 pg/mL if your target protein concentration is predicted to be extremely high.

Quality Control Requirements

For reliable quantification, ensure your standard curve meets rigorous quality criteria. The R² value of your standard curve trend line should exceed 0.99 to indicate proper antibody binding and protein capture. Additionally, when testing samples in complex matrices (such as serum or cell lysates), include spike controls—standards diluted in the same matrix as your samples—to verify that the target protein remains recoverable and to detect any matrix interference effects.

Data Analysis

After generating your standard curve with the recombinant DCC protein, use appropriate curve-fitting algorithms (such as 4-parameter models) for data analysis, subtract background absorbance, account for sample dilutions, and calculate statistical measures including average, standard deviation, and coefficient of variation.

Recombinant Mouse DCC has been validated for several key applications in published research, including immunohistochemistry (IHC), Western blotting, immunoprecipitation, receptor-ligand blockade, and neutralization assays.

Essential validated applications:

• Immunohistochemistry (IHC): Used to detect DCC protein localization in mouse brain and neural tissues, including paraffin-embedded and frozen sections. Specific staining has been demonstrated in neuronal processes and commissural axons.
• Western Blot: Validated for detection of DCC protein in tissue homogenates and cell lysates, confirming protein expression and molecular weight.
• Immunoprecipitation: Applied to isolate DCC from tissue homogenates for downstream analysis, such as protein-protein interaction studies.
• Blockade of receptor-ligand interaction: Used in functional assays to study DCC’s interaction with netrin-1 and its role in axon guidance and apoptosis.
• Neutralization: Employed to inhibit DCC function in cell-based assays, such as studies on oligodendrocyte precursor migration and proliferation.

Additional context from published research:

• DCC has been studied in the context of neural development, axon guidance, and apoptosis, with applications in both embryonic and adult mouse tissues.
• Functional studies have used recombinant DCC to investigate its role in opioid maladaptation, depression-like behaviors, and astroglial development.
• The protein has also been used in genetic and pharmacological models to dissect its involvement in disease phenotypes and neural circuit formation.

Summary Table: Validated Applications for Recombinant Mouse DCC

These applications are supported by multiple peer-reviewed studies and product datasheets, confirming their reliability for research involving mouse DCC protein.

To reconstitute and prepare Recombinant Mouse DCC protein for cell culture experiments, use sterile technique and follow these steps:

• Reconstitution: Add sterile phosphate-buffered saline (PBS) to the lyophilized protein to achieve the desired concentration, commonly 200 μg/mL. If the product datasheet specifies a different buffer or concentration, follow those instructions.
• Mixing: Gently swirl or invert the vial to dissolve the protein. Avoid vigorous shaking or vortexing, as this can cause foaming and protein denaturation.
• Incubation: Allow the solution to sit at room temperature for 15–30 minutes to ensure complete dissolution.
• Aliquoting: If not using immediately, aliquot the reconstituted protein into single-use volumes to avoid repeated freeze-thaw cycles, which can degrade protein activity.
• Storage: Store aliquots at −20°C to −70°C for long-term storage, or at 2–8°C for up to one month if you plan to use them soon. Avoid repeated freeze-thaw cycles.
• Sterility: If sterility is critical for your application, filter the reconstituted protein through a 0.2 μm or 0.22 μm filter before use.
• Dilution for Cell Culture: For cell culture, dilute the reconstituted protein into your culture medium. It is recommended to use medium containing 0.5% BSA or 5% fetal calf serum to minimize non-specific adsorption and maintain protein stability.

Key points for cell culture use:

• Always use aseptic technique.
• Prepare working dilutions fresh before each experiment.
• If the protein is sensitive to adsorption, minimize time in plasticware and use carrier proteins (BSA or serum) in dilutions.
• Confirm the final buffer composition is compatible with your cells (e.g., PBS without calcium or magnesium, pH 7.2–7.3).

Summary protocol:

1. Add sterile PBS (or specified buffer) to lyophilized DCC protein to reach 200 μg/mL.
2. Gently mix and incubate at room temperature for 15–30 minutes.
3. Aliquot and store at −20°C to −70°C.
4. For cell culture, dilute in medium with 0.5% BSA or 5% FCS immediately before use.

These steps will ensure optimal recovery, stability, and activity of recombinant Mouse DCC protein for cell culture experiments.