Recombinant Human Myostatin Propeptide

Using Recombinant Human Myostatin Propeptide in research applications is valuable for studies aiming to enhance muscle growth, promote muscle and bone regeneration, or investigate the inhibition of myostatin signaling. This propeptide acts as a potent and specific inhibitor of myostatin (GDF-8), a negative regulator of skeletal muscle mass and a modulator of bone metabolism.

Key scientific reasons to use this reagent include:

• Muscle Growth Enhancement: The recombinant propeptide binds to and inhibits myostatin, leading to significant increases in skeletal muscle mass. In animal models, administration of the propeptide resulted in up to a 20% increase in muscle mass and up to 200% in cases of overexpression, mimicking the phenotype of myostatin knockout animals.
• Muscle and Bone Repair: Blocking myostatin with the propeptide accelerates muscle regeneration and improves bone healing. For example, in a mouse injury model, treatment with recombinant myostatin propeptide increased muscle mass, enhanced bone callus formation, and improved bone volume at the injury site.
• Disease Modeling and Therapeutic Research: The propeptide is used to model and potentially treat muscle-wasting diseases (e.g., muscular dystrophies, sarcopenia, spinal muscular atrophy) by promoting muscle hypertrophy and reducing fibrosis during muscle repair.
• Mechanistic Studies: It is a tool for dissecting the TGF-β signaling pathway, particularly the regulation of the Akt/mTOR pathway, which is central to protein synthesis and muscle growth.
• High Specificity and Potency: The recombinant propeptide exhibits high binding affinity for myostatin, ensuring effective inhibition with minimal off-target effects.

Typical applications include:

• In vitro and in vivo studies of muscle development, regeneration, and atrophy.
• Bone healing and musculoskeletal tissue engineering research.
• Preclinical evaluation of myostatin inhibition as a therapeutic strategy for muscle and bone disorders.

Summary:
Recombinant Human Myostatin Propeptide is a well-characterized, potent inhibitor of myostatin, making it a critical reagent for research focused on muscle and bone growth, regeneration, and disease modeling where modulation of myostatin activity is desired.

Recombinant Human Myostatin Propeptide can be used as a standard for quantification or calibration in ELISA assays only if your assay is specifically designed to detect the propeptide form, not the mature myostatin protein. The suitability depends on the specificity of the antibodies used in your ELISA and the form of myostatin you intend to quantify.

Key considerations:

• Protein Form: The recombinant human myostatin propeptide is a fragment corresponding to the N-terminal propeptide region, not the mature, biologically active C-terminal dimer of myostatin. Most commercial ELISA kits for myostatin are designed to detect the mature form (GDF-8, aa 268–376), not the propeptide. If your ELISA is designed for the mature myostatin, using the propeptide as a standard will result in inaccurate quantification.

• Antibody Specificity: If your ELISA uses antibodies that specifically recognize the propeptide region, then the recombinant propeptide is appropriate as a standard. However, if the antibodies are specific for the mature myostatin or the latent complex, the propeptide will not be detected, or the response will not be equivalent.

• Assay Validation: For accurate quantification, the standard must be immunologically equivalent to the analyte in your samples. Dose-response curves for the standard and the endogenous analyte should be parallel, and recovery/linearity should be validated.

• Manufacturer Recommendations: Some ELISA kit manufacturers explicitly state that their kits are not recommended for detection of recombinant proteins unless the recombinant standard matches the target analyte in sequence and conformation.

Summary Table:

Best Practice:

• Check your ELISA kit documentation for the target epitope and recommended standard.
• Validate that the recombinant propeptide produces a standard curve parallel to your sample analyte.
• Do not use the propeptide as a standard for mature myostatin quantification unless the assay is specifically validated for this purpose.

If you are developing a custom ELISA for the propeptide, recombinant human myostatin propeptide is suitable as a standard, provided you validate its performance in your assay system. If you are using a commercial kit for mature myostatin, use the recommended mature myostatin standard.

Recombinant Human Myostatin Propeptide has been validated in published research for several key applications, primarily focused on inhibition of myostatin activity, enhancement of muscle growth, and promotion of musculoskeletal repair and regeneration.

Validated Applications in Published Research:

• Muscle Growth Enhancement:
  Systemic administration of recombinant myostatin propeptide in animal models (mice) has been shown to significantly increase skeletal muscle mass (by up to 20%) through myofiber hypertrophy, without affecting cardiac muscle or causing organomegaly. This effect is due to the propeptide’s ability to bind and inhibit active myostatin, a negative regulator of muscle development.

• Muscle and Bone Repair/Regeneration:
  In models of deep musculoskeletal injury (e.g., fibula osteotomy with muscle damage), recombinant myostatin propeptide treatment accelerated both muscle and bone healing. Treated animals exhibited increased muscle mass and improved bone bridging at the injury site, as assessed by micro-CT and histological analysis. These findings support its use in research on orthopedic trauma and extremity injury.

• Functional Assays (In Vitro and In Vivo):
  The propeptide has been validated for use in functional assays to study myostatin inhibition, including cell-based bioactivity assays and animal studies measuring muscle hypertrophy and tissue regeneration. It is used to confirm its ability to block myostatin signaling and promote anabolic effects.

• Detection and Pharmacokinetics:
  Research has investigated the detectability and metabolism of recombinant myostatin propeptide in serum and urine following administration in animal models, relevant for anti-doping studies and pharmacokinetic profiling.

Additional Context:

• The recombinant propeptide is typically expressed in E. coli and purified for use in both cell culture and animal studies.
• Its mechanism involves binding to myostatin and preventing its activation, thereby lifting the inhibition on muscle growth and repair processes.
• Applications extend to potential therapeutic research for muscle-wasting diseases, muscular dystrophy, and bone healing, though most published validations are preclinical.

Summary Table of Validated Applications

These applications are supported by multiple peer-reviewed studies and are considered validated for research use in the contexts described above.

To reconstitute and prepare Recombinant Human Myostatin Propeptide for cell culture experiments, dissolve the lyophilized protein in sterile 20 mM HCl at a concentration of 0.1 mg/mL; this stock can then be further diluted into your desired cell culture medium or buffer.

Step-by-step protocol:

1. Centrifuge the vial briefly before opening to ensure all powder is at the bottom.
2. Add sterile 20 mM HCl to the vial to achieve a final concentration of 0.1 mg/mL.
3. Gently pipette up and down or swirl to dissolve the protein completely; avoid vigorous shaking or foaming.
4. Aliquot the solution into working volumes to avoid repeated freeze-thaw cycles.
5. For long-term storage, add a carrier protein such as 0.1% BSA or HSA to the aliquots, then store at –20°C or –80°C. For short-term use (up to one week), store at 4°C.
6. Before use in cell culture, dilute the stock solution into your cell culture medium. If your experiment is serum-free or animal protein-free, avoid BSA/HSA and consider using trehalose as a stabilizer.
7. Always filter sterilize the final working solution if sterility is required and the protein is not supplied sterile.

Additional notes:

• If the product datasheet recommends a different buffer (e.g., water or Tris), follow the manufacturer’s instructions, but 20 mM HCl at 0.1 mg/mL is a widely accepted protocol for this protein.
• Avoid repeated freeze-thaw cycles, as this can degrade the protein.
• For best results, consult the Certificate of Analysis (CoA) provided with your specific lot for any lot-specific recommendations.

Summary of key points:

• Reconstitution buffer: Sterile 20 mM HCl
• Stock concentration: 0.1 mg/mL
• Aliquot and storage: –20°C or –80°C with carrier protein for long-term; 4°C for short-term
• Dilution: Directly into cell culture medium before use

This protocol ensures optimal solubility and stability of the recombinant myostatin propeptide for cell culture applications.