Recombinant Mouse Sonic Hedgehog (N Terminus)

Recombinant Mouse Sonic Hedgehog (N Terminus) is used in research applications because it is the biologically active fragment responsible for mediating key signaling events in embryonic development, stem cell differentiation, tissue regeneration, and disease modeling. This fragment is essential for activating the Hedgehog signaling pathway, which regulates cell fate, proliferation, and patterning in multiple tissues.

Key scientific reasons to use this reagent include:

• Biological Activity: The N-terminal fragment (Shh-N) is the active signaling domain generated by autocatalytic cleavage of the full-length Sonic Hedgehog protein. It binds to the Patched (PTCH1/2) and Smoothened (SMO) receptors, initiating downstream signaling critical for neural, limb, and somite patterning during embryogenesis.
• Stem Cell and Developmental Biology: Shh-N is widely used to direct the differentiation of embryonic stem cells, neural progenitors, and mesenchymal stem cells, and to study ventral neural tube and limb development. It is also used to induce specific cell fates in vitro, such as dopaminergic neurons for Parkinson’s disease models.
• Tissue Regeneration and Disease Modeling: Shh-N is involved in tissue repair, regeneration, and cancer biology. It is used to study mechanisms of tissue remodeling, stem cell maintenance, and tumorigenesis.
• Validated Bioactivity: Recombinant Shh-N is quality-tested for bioactivity, such as its ability to induce differentiation in C3H10T1/2 cells, ensuring reproducible results in functional assays.
• Conservation and Relevance: The N-terminal domain is highly conserved across species, making mouse Shh-N suitable for cross-species studies and translational research.

Typical applications include:

• Patterning and differentiation of neural and mesodermal tissues in stem cell cultures.
• Functional studies of Hedgehog pathway activation or inhibition in development, regeneration, and cancer.
• Use as a standard in ELISA or as a positive control in bioassays.

Technical considerations:

• The recombinant N-terminal fragment is typically supplied at high purity and validated for activity, with or without carrier proteins depending on the application.
• Lipid modifications of Shh-N enhance its receptor interactions and biological potency, which is relevant for in vitro and in vivo studies.

In summary, Recombinant Mouse Sonic Hedgehog (N Terminus) is the preferred tool for activating the Hedgehog pathway in controlled experimental systems, enabling precise studies of development, regeneration, and disease mechanisms.

You can use recombinant Mouse Sonic Hedgehog (N Terminus) as a standard for quantification or calibration in ELISA assays, provided that the recombinant protein is of high purity, its concentration is accurately determined, and it is compatible with the antibodies and detection system used in your specific ELISA setup.

Key considerations:

• Purity and Characterization: The recombinant protein should be highly purified (≥95% purity is typical) and well-characterized, with a defined concentration. This ensures that the standard curve reflects the true concentration of the analyte.
• Form and Sequence: The recombinant standard should match the form (e.g., N-terminal fragment) and sequence of the endogenous protein detected by your ELISA antibodies. Most commercial ELISA kits for mouse Shh-N use recombinant Shh-N as the standard.
• Matrix Effects: Prepare the standard curve in the same buffer or matrix as your samples to minimize matrix effects and ensure accurate quantification.
• Validation: If you are using a recombinant standard not supplied with a commercial ELISA kit, you should validate its performance in your assay. This includes confirming parallelism between the standard curve and serially diluted biological samples to ensure accurate quantification.

Best Practices:

• Reconstitution and Handling: Follow manufacturer or supplier instructions for reconstitution and storage to maintain protein integrity.
• Concentration Determination: Use precise methods (e.g., absorbance at 280 nm, BCA assay) to determine the concentration of your recombinant standard.
• Standard Curve Range: Choose a standard curve range that encompasses the expected concentrations in your samples, typically from low pg/mL to ng/mL for Shh-N ELISAs.

Caveats:

• Some recombinant proteins are specifically labeled for use as ELISA standards and are tested for this application, while others are intended for bioassays or functional studies and may not be validated for ELISA calibration. Always check the product documentation or consult the supplier if uncertain.
• If your ELISA kit provides its own standard, it is best to use the supplied standard for optimal accuracy, as it is validated for that specific assay.

In summary, recombinant Mouse Sonic Hedgehog (N Terminus) can be used as an ELISA standard if it is highly purified, accurately quantified, and validated for compatibility with your assay system.

Recombinant Mouse Sonic Hedgehog (N Terminus) has been validated in published research for several key applications, primarily involving bioassays, cell culture, differentiation studies, in vivo experiments, and tissue culture.

Validated Applications in Published Research:

• Bioassay: Used to assess biological activity, such as induction of alkaline phosphatase in C3H10T1/2 mouse embryonic fibroblast cells, and to study signaling pathway activation in various cell types.
• Cell Culture: Applied to maintain or differentiate stem cells, including neural progenitors and dopaminergic neurons derived from pluripotent stem cells.
• Differentiation: Facilitates differentiation of stem cells into specific lineages, such as ventral midbrain dopaminergic neurons, oligodendrocyte progenitors, and other neural cell types.
• In Vivo: Used in animal models to study developmental processes, tissue regeneration, and disease mechanisms, including spinal cord injury, Down syndrome models, and Parkinson’s disease.
• Tissue Culture: Supports tissue explant cultures for morphogenesis and developmental biology studies.

Representative Published Research Applications:

• Neural Development and Regeneration: Promotes myelin regeneration and oligodendrocyte progenitor proliferation in spinal cord injury models.
• Stem Cell Differentiation: Induces differentiation of human and mouse pluripotent stem cells into dopaminergic neurons and other neural lineages.
• Cancer and Disease Models: Investigates Hedgehog pathway roles in cancer development, tissue remodeling, and neurodegeneration.
• Morphogenesis: Regulates developmental morphogenesis of the central nervous system, limb, and other tissues.
• Cell Signaling Studies: Used to dissect Hedgehog pathway interactions with Notch, Wnt, and BMP signaling in vitro and in vivo.

Additional Validated Techniques:

• ELISA: Used as a standard for quantifying Sonic Hedgehog protein levels in biological samples.
• Western Blot: Detection and quantification of Shh protein in cell and tissue lysates.
• 3D Cell Culture and Organoid Models: Supports maturation and integration of brain organoids and other complex tissue models.

Summary Table of Applications

These applications are supported by numerous peer-reviewed publications, demonstrating the protein’s utility in both basic and translational research across developmental biology, neuroscience, regenerative medicine, and disease modeling.

To reconstitute and prepare Recombinant Mouse Sonic Hedgehog (N Terminus) protein for cell culture experiments, follow these steps:

• Reconstitution:

  • For proteins supplied with carrier protein (e.g., BSA): Reconstitute the lyophilized protein at 100 μg/mL in sterile PBS containing at least 0.1% human or bovine serum albumin.
  • For carrier-free formulations: Reconstitute at 100 μg/mL in sterile PBS.
  • Alternatively, some protocols allow reconstitution in sterile distilled water to a concentration of 0.1–0.5 mg/mL. However, PBS with carrier protein is preferred for stability in cell culture applications.

• Aliquoting and Storage:

  • After reconstitution, aliquot the stock solution to avoid repeated freeze-thaw cycles.
  • Store aliquots at –20 °C to –70 °C for up to 3 months under sterile conditions.
  • For short-term use (up to 1 month), storage at 2–8 °C is acceptable.

• Working Solution Preparation:

  • Before adding to cell cultures, dilute the stock solution to the desired working concentration using cell culture medium or buffered solution containing carrier protein (e.g., PBS with 0.1% BSA).
  • The effective working concentration for bioactivity (e.g., induction of alkaline phosphatase in C3H10T1/2 cells) is typically in the range of 0.6–3 μg/mL, but this should be optimized for your specific assay.

• General Best Practices:

  • Use sterile technique throughout to prevent contamination.
  • Avoid repeated freeze-thaw cycles, as this can reduce protein activity and stability.
  • If using a carrier-free formulation and stability is a concern, consider adding a carrier protein (e.g., BSA) to working solutions.

Summary Table: Reconstitution and Storage

References:

If you need protocol details for a specific cell type or assay, please specify for tailored recommendations.