Recombinant Human Sonic Hedgehog (C24II) N-Terminus

Recombinant Human Sonic Hedgehog (C24II) N-Terminus is widely used in research because it provides a highly active, standardized form of the SHH morphogen, essential for studies involving cell differentiation, embryonic development, and stem cell biology.

Key scientific reasons to use this reagent include:

• Enhanced Biological Activity: The C24II variant contains a Cys24Ile-Ile substitution in the N-terminal domain, which increases its lipophilicity and bioactivity compared to wild-type SHH-N. This modification results in more potent signaling, allowing for lower effective concentrations in bioassays and cell culture applications.

• Critical Role in Developmental Biology: SHH is a central morphogen in embryogenesis, regulating patterning of the central nervous system, somites, limbs, and other tissues. It is indispensable for studies on neurogenesis, limb development, hematopoiesis, and organogenesis.

• Stem Cell Differentiation and Maintenance: Recombinant SHH (C24II) is routinely used to promote the growth and differentiation of embryonic stem cells, mesenchymal stem cells, and neural precursors. It is essential for protocols aiming to generate specific cell types, such as dopaminergic neurons or ameloblasts, from pluripotent stem cells.

• Functional Assays and Disease Modeling: The protein is validated for inducing alkaline phosphatase production in C3H10T1/2 cells, a standard assay for SHH activity. It is also used in cancer research, tissue regeneration studies, and for investigating Hedgehog pathway involvement in diseases such as medulloblastoma and neuroendocrine tumors.

• High Purity and Consistency: Research-grade recombinant SHH (C24II) is typically supplied at >95% purity, with low endotoxin levels, ensuring reproducibility and reliability in sensitive cell culture and differentiation experiments.

• Versatility in Applications: It is suitable for a wide range of applications, including:

  • Expansion and differentiation of human and mouse stem cells
  • Functional bioassays (e.g., alkaline phosphatase induction)
  • Neural lineage specification
  • Cancer pathway studies
  • Tissue engineering and regenerative medicine

Summary Table: Key Features of Recombinant Human SHH (C24II) N-Terminus

In summary, Recombinant Human SHH (C24II) N-Terminus is a preferred tool for developmental biology, stem cell research, and disease modeling due to its enhanced activity, reliability, and broad applicability in functional and differentiation assays.

Yes, Recombinant Human Sonic Hedgehog (C24II) N-Terminus can be used as a standard for quantification or calibration in ELISA assays, provided it is of high purity and properly formulated. Multiple sources recommend using recombinant proteins, including this specific SHH variant, as ELISA standards, especially when supplied with BSA as a carrier for stability.

Key considerations and supporting details:

• Formulation: It is generally advised to use the recombinant protein with BSA for ELISA standards, as BSA helps stabilize the protein and prevents adsorption to plasticware. Carrier-free formulations are also available but may require additional precautions to prevent protein loss.

• Purity and Quality: The protein should be highly pure (typically >95% by SDS-PAGE) and free from significant contaminants such as endotoxin, host cell proteins, and DNA. The referenced SHH (C24II) N-Terminus meets these criteria, with low endotoxin and high purity.

• Bioactivity: The recombinant SHH (C24II) N-Terminus is bioactive and has been validated in functional assays (e.g., induction of alkaline phosphatase in C3H10T1/2 cells), indicating correct folding and activity.

• ELISA Compatibility: Vendors and technical resources explicitly state that this recombinant protein is suitable for use as an ELISA standard. General ELISA guidelines also support the use of purified recombinant proteins as standards for generating standard curves.

• Reconstitution: Follow the manufacturer’s instructions for reconstitution, typically in PBS with at least 0.1% BSA, and allow sufficient time for complete solubilization.

• Standard Curve Preparation: Prepare serial dilutions from the reconstituted stock to generate a standard curve covering the expected range of your samples.

Additional notes:

• Validation: If you are using a commercial ELISA kit, ensure that the antibody pair in the kit recognizes the same SHH (C24II) N-terminal epitope as your standard. Some kits are validated with E. coli-expressed SHH-N, which is structurally similar to the C24II variant.

• Documentation: For regulated or clinical applications, ensure the recombinant standard is produced under appropriate quality guidelines (e.g., GMP) and that you retain certificates of analysis for traceability.

In summary, Recombinant Human Sonic Hedgehog (C24II) N-Terminus is suitable as an ELISA standard if it is of high purity, properly formulated, and recognized by the assay antibodies. Always validate the standard curve in your specific assay context.

Recombinant Human Sonic Hedgehog (C24II) N-Terminus has been validated primarily for bioactivity assays in published research, with applications focused on functional studies of Hedgehog signaling, cell differentiation, proliferation, and stem cell biology.

Key validated applications in published research include:

• Bioassays/Functional Activity:
  The protein is widely used to activate Hedgehog signaling in cell-based bioassays, including induction of alkaline phosphatase in C3H10T1/2 mouse embryonic fibroblast cells, a standard readout for Hedgehog pathway activation.
  It has been used to assess pathway activation in various cell types, including human pluripotent stem cells, neuronal cells, and cancer cell lines.

• Stem Cell Differentiation and Maintenance:
  Validated for promoting the growth and differentiation of embryonic stem cells, mesenchymal stem cells, and neuronal lineage cells in vitro.
  Used in protocols for midbrain specification, ameloblast induction, and neural differentiation from human pluripotent stem cells.

• Cell Proliferation and Survival:
  Applied in studies investigating proliferation and survival of neural progenitors, cancer cells, and other cell types responsive to Hedgehog signaling.

• In Vivo Studies:
  Used in animal models to study the effects of Hedgehog pathway activation on tissue development, regeneration, and disease models, including neurodegenerative and cancer models.

• Cancer Research:
  Utilized to probe Hedgehog pathway roles in tumorigenesis, including medulloblastoma, neuroendocrine lung cancer, and breast cancer, often in combination with pathway inhibitors to assess functional outcomes.

• Protein-Protein Interaction and Binding Assays:
  Employed in binding assays to study interactions with Hedgehog pathway receptors and co-factors.

• ELISA Standard:
  Used as a standard in ELISA assays for quantifying Hedgehog pathway components.

Summary Table of Validated Applications

Species and Sample Types:
Most published studies use human or mouse cells (whole cells, transfected cells, stem cells) for in vitro assays, with some in vivo validation in animal models.

Notes on Protocols:

• The protein is often used in serum-free or defined media for cell culture and differentiation protocols.
• Activity is typically confirmed by induction of downstream Hedgehog target genes or reporter assays.

Conclusion:
The primary validated application for Recombinant Human Sonic Hedgehog (C24II) N-Terminus in published research is as a bioactive reagent for functional Hedgehog pathway studies, especially in stem cell biology, developmental biology, and cancer research.

To reconstitute and prepare Recombinant Human Sonic Hedgehog (C24II) N-Terminus protein for cell culture experiments, dissolve the lyophilized protein at 100–200 μg/mL in sterile PBS containing at least 0.1% human or bovine serum albumin (BSA or HSA), and allow up to 24 hours at 2–8 °C for complete reconstitution.

Detailed protocol and best practices:

• Reconstitution:

  • Add sterile PBS (phosphate-buffered saline) to the lyophilized protein to achieve a final concentration of 100–200 μg/mL.
  • Include at least 0.1% BSA or HSA in the PBS to stabilize the protein and prevent adsorption to tube surfaces.
  • Gently mix by inversion or slow pipetting; avoid vigorous vortexing to prevent protein denaturation.
  • Incubate the solution at 2–8 °C (refrigerator) for up to 24 hours to ensure complete dissolution.

• Aliquoting and Storage:

  • After reconstitution, aliquot the solution to avoid repeated freeze-thaw cycles, which can degrade the protein.
  • Store aliquots at 2–8 °C for up to 1 month, or at ≤ –20 °C for up to 3 months under sterile conditions.
  • For short-term use (within 2–7 days), storage at 4–8 °C is acceptable.
  • Avoid multiple freeze-thaw cycles by using single-use aliquots.

• Preparation for Cell Culture:

  • Before adding to cell cultures, dilute the reconstituted stock to the desired working concentration using cell culture medium supplemented with 0.1% BSA or HSA to maintain protein stability.
  • Typical working concentrations for bioassays range from 0.1–0.4 μg/mL, but optimal dosing should be empirically determined for your specific application.

• Sterility:

  • Ensure all solutions and materials are sterile to prevent contamination of cell cultures.
  • If necessary, filter the reconstituted protein solution through a 0.2 μm sterile filter.

Summary Table:

Additional notes:

• Always consult the specific product datasheet for any lot-specific recommendations.
• If the protein is supplied without carrier protein (BSA/HSA), it is especially important to add it during reconstitution to prevent loss of activity due to adsorption.
• For sensitive applications, confirm protein activity with a functional assay before use.

This protocol ensures optimal stability and bioactivity of recombinant SHH (C24II) N-terminus for cell culture experiments.