Recombinant Human Myostatin

Recombinant Human Myostatin is used in research applications to study and manipulate muscle growth, metabolism, and bone repair due to its role as a potent negative regulator of skeletal muscle mass and its involvement in various signaling pathways.

Key scientific applications and rationale:

• Muscle Growth Regulation: Myostatin (GDF-8) inhibits muscle cell proliferation and differentiation. Using recombinant human myostatin allows researchers to investigate mechanisms of muscle development, atrophy, and hypertrophy, and to model muscle-wasting diseases such as muscular dystrophy and sarcopenia.

• Bone and Musculoskeletal Repair: Recombinant myostatin and its inhibitors (e.g., propeptide) have been shown to enhance muscle and bone regeneration in injury models. Blocking myostatin function can significantly improve repair outcomes, making recombinant myostatin valuable for studying musculoskeletal tissue regeneration.

• Metabolic Studies: Myostatin affects lipid metabolism and energy homeostasis. Recombinant protein is used to analyze its impact on adipocyte differentiation, insulin sensitivity, and metabolic disorders.

• Cell Signaling and Pathway Analysis: Recombinant myostatin is essential for dissecting TGF-β superfamily signaling, including its effects on downstream targets such as RANKL, sclerostin, and Dkk1 in bone cells, and for understanding its role in WNT/β-catenin pathway inhibition.

• Bioassays and Functional Studies: Recombinant human myostatin is used in cell culture, bioassays, and in vivo models to quantify biological activity, receptor interactions, and to screen for inhibitors or therapeutic candidates.

• ELISA and Western Blot Standards: It serves as a standard or control in immunoassays for quantifying endogenous myostatin levels in biological samples.

Best practices:

• Use recombinant human myostatin at concentrations validated for your specific assay (e.g., bioactivity, binding studies).
• Confirm purity and activity via SDS-PAGE, HPLC, or functional assays.
• For inhibition studies, pair with recombinant myostatin propeptide or specific antibodies to dissect pathway effects.

Summary:
Recombinant human myostatin is a critical tool for research into muscle biology, metabolic regulation, bone health, and therapeutic development for muscle-wasting conditions. Its use enables precise mechanistic studies and the development of interventions targeting myostatin signaling.

You can use recombinant human myostatin as a standard for quantification or calibration in ELISA assays, but only if the ELISA kit is validated to recognize and quantify recombinant myostatin. The suitability depends on the specific ELISA kit and its antibody specificity.

Key considerations:

• Kit Validation: Some ELISA kits are specifically validated to detect both natural and recombinant human myostatin. For example, certain kits state: "The assay will exclusively recognize both natural and recombinant human GDF-8/Myostatin". Another manufacturer's documentation confirms: "This immunoassay has been shown to quantitate the recombinant GDF-8 accurately. Results obtained using natural GDF-8 showed dose-response curves that were parallel to the standard curves obtained using the Quantikine® kit standards". This means recombinant myostatin can be reliably used as a standard in these assays.

• Antibody Specificity: Some ELISA kits are designed to detect only native (endogenous) myostatin and may not recognize recombinant forms, especially if the recombinant protein differs in post-translational modifications, folding, or sequence (e.g., tags, truncations). These kits often state: "The ELISA Kit is designed to detect native, not recombinant, MYOSTATIN". Using recombinant myostatin as a standard in such kits could lead to inaccurate quantification.

• Calibration and Parallelism: For accurate quantification, the standard curve generated with recombinant myostatin must be parallel to the response obtained with native myostatin in your sample matrix. Some kits provide data showing parallelism between recombinant and native forms, supporting their interchangeability as standards.

• Expression System and Purity: Recombinant myostatin can be produced in different systems (e.g., E. coli, mammalian cells), which may affect its structure and immunoreactivity. Ensure the recombinant standard matches the form recognized by your assay antibodies.

Best Practices:

• Check your ELISA kit documentation for explicit statements about compatibility with recombinant standards.
• If not specified, perform a parallelism test: spike known amounts of recombinant myostatin into your sample matrix and verify that the standard curve is parallel to the endogenous signal.
• Use a recombinant standard that matches the mature, active form of myostatin, as recognized by your assay.

Summary Table: Recombinant Myostatin as ELISA Standard

In conclusion:
You can use recombinant human myostatin as a standard for ELISA quantification only if your kit is validated for it or you have experimentally confirmed parallelism between recombinant and native forms in your assay system. Always consult your kit's technical manual and, if necessary, perform validation experiments.

Recombinant Human Myostatin has been validated for a range of applications in published research, primarily in studies of muscle growth regulation, musculoskeletal repair, metabolic regulation, and assay development.

Key validated applications include:

• Functional/Bioassays:
  Recombinant human myostatin is widely used in in vitro and in vivo bioassays to study its effects on muscle cell differentiation, proliferation, and hypertrophy, as well as its inhibitory role in muscle growth. For example, it has been used to induce hemoglobin expression in K562 cells and to assess its biological activity in various cell-based assays.

• Muscle and Bone Regeneration Models:
  In animal models, recombinant myostatin (including propeptide forms) has been validated for enhancing or inhibiting muscle and bone repair. For instance, systemic administration of recombinant myostatin propeptide in mice improved muscle and bone healing following injury, as assessed by microCT, histology, and muscle mass measurements. These studies often use recombinant myostatin to model or counteract muscle wasting and to test therapeutic interventions.

• Metabolic Studies:
  Recombinant myostatin has been used to investigate its role in lipid metabolism and adipocyte differentiation, demonstrating its inhibitory effects on preadipocyte differentiation and influence on lipid catabolism.

• Binding and Interaction Assays:
  It has been validated in binding assays, such as surface plasmon resonance, to study interactions with receptors, inhibitors (e.g., follistatin), and other proteins involved in the TGF-β signaling pathway.

• ELISA and Immunoassay Development:
  Recombinant human myostatin is used as a standard or control in ELISA and other immunoassays for quantifying myostatin levels in biological samples, supporting both research and clinical assay development.

• In Vivo Studies:
  Recombinant myostatin and its inhibitors have been used in in vivo studies to assess effects on muscle mass, strength, and regeneration, particularly in models of muscular dystrophy and muscle injury.

• Cell Culture:
  It is used to treat cultured cells (e.g., myoblasts, adipocytes) to study signaling pathways, gene expression, and cellular responses to myostatin.

Summary Table of Validated Applications

These applications are supported by both primary research articles and product validation data from scientific suppliers, reflecting broad utility in muscle biology, regenerative medicine, and metabolic research.

To reconstitute and prepare Recombinant Human Myostatin protein for cell culture experiments, dissolve the lyophilized protein in sterile acidic buffer—commonly 4–20 mM HCl—at a concentration of 0.1–0.5 mg/mL, optionally with 0.1% carrier protein (such as BSA or HSA) to enhance stability and prevent adsorption to surfaces.

Step-by-step protocol:

1. Equilibrate and Centrifuge:

   • Allow the vial to reach room temperature before opening to prevent condensation.
   • Briefly centrifuge the vial (20–30 seconds) to collect all powder at the bottom.

2. Reconstitution:

   • Add sterile 4 mM HCl (or up to 20 mM HCl, depending on the specific product) to achieve a final concentration of 0.1–0.5 mg/mL.
   • For enhanced stability, especially for long-term storage or low-concentration use, include at least 0.1% serum albumin (BSA or HSA) in the reconstitution buffer.
   • Gently mix by pipetting or slow inversion. Do not vortex, as this may denature the protein.

3. Solubilization:

   • Allow the solution to sit at room temperature for 15–30 minutes with gentle agitation. If undissolved material remains, extend mixing up to 2 hours.
   • If solubility issues persist, incubate overnight at 4°C.

4. Aliquot and Storage:

   • Aliquot the reconstituted protein to avoid repeated freeze-thaw cycles, which can cause denaturation.
   • Store aliquots at 4°C for up to 1 month, or at –20°C to –80°C for longer-term storage.
   • For cell culture, further dilute the stock solution into cell culture medium or assay buffer immediately before use.

5. Working Concentration:

   • Typical working concentrations for cell culture experiments are in the range of 2–25 ng/mL, depending on the assay and cell type.

Additional notes:

• Always consult the specific product datasheet for any unique requirements, as some preparations may differ in formulation or recommended buffer.
• Avoid reconstituting at concentrations above 1 mg/mL to prevent aggregation.
• Confirm protein integrity and concentration by SDS-PAGE or BCA assay if needed.

Summary Table:

This protocol ensures maximum solubility, stability, and bioactivity of recombinant human myostatin for cell culture applications.