Recombinant Human β-NGF

Recombinant Human β-NGF is widely used in research applications due to its critical role in the development, maintenance, and regeneration of sensory and sympathetic neurons, as well as its broader effects on tissue repair and immune modulation.

Key scientific reasons to use recombinant human β-NGF:

• Neurotrophic Activity: β-NGF is essential for the survival, growth, and differentiation of neural cells, making it a fundamental tool for studies on neuronal development, neurodegeneration, and regeneration.
• Modeling Neurodegenerative Diseases: Recombinant β-NGF is used to prevent neuronal degeneration in animal models and is a therapeutic target in neuron regeneration studies, supporting research into conditions such as Alzheimer’s disease, diabetic neuropathy, and HIV-associated sensory neuropathy.
• Bone and Tissue Repair: β-NGF promotes endochondral ossification and accelerates fracture healing by stimulating osteogenesis, increasing bone mineral density, and improving bone architecture in animal models. It upregulates genes involved in cartilage maturation and tissue mineralization, making it valuable for musculoskeletal research.
• Immune System Modulation: β-NGF acts as a pleiotropic cytokine, influencing mast cell proliferation, histamine release, B lymphocyte growth, and neutrophil survival, thus supporting studies on neuro-immune interactions and inflammation.
• Cellular Differentiation and Bioassays: Recombinant β-NGF induces differentiation of dorsal root ganglion (DRG) and PC12 cells, and activates TrkA receptor phosphorylation, serving as a reliable positive control in bioassays and cell signaling studies.
• Reproducibility and Scalability: Recombinant production ensures batch-to-batch consistency, eliminates animal-derived contaminants, and enables large-scale use for both basic research and preclinical studies.

Typical research applications include:

• Neural cell culture and differentiation protocols
• Disease modeling for neurodegeneration and neuropathies
• Bone and cartilage tissue engineering
• Immune cell functional assays
• Screening for neuroprotective or regenerative therapeutics

In summary, recombinant human β-NGF is a versatile and essential reagent for neuroscience, tissue engineering, and immunology research, providing reproducible and biologically active material for mechanistic studies and therapeutic development.

You can use recombinant human β-NGF as a standard for quantification or calibration in your ELISA assays, provided that the recombinant protein is of high purity, its concentration is accurately known, and it is compatible with the antibodies used in your assay.

Key considerations:

• Recombinant β-NGF is widely used as an ELISA standard: Many commercial ELISA kits for human β-NGF quantification use recombinant human β-NGF as their standard, often produced in mammalian cells for proper folding and post-translational modifications.
• Standard curve generation: The recombinant standard should be serially diluted to generate a standard curve within the validated range of your ELISA kit (e.g., 3.9–5000 pg/mL, depending on the kit).
• Parallelism: For accurate quantification, the recombinant standard should behave similarly to endogenous β-NGF in your sample matrix. Most kits validate that their recombinant standard and natural protein are recognized equivalently by the assay antibodies.
• Source and validation: Ideally, use a recombinant β-NGF standard that is referenced or validated by a recognized authority (e.g., WHO reference reagent) or is specifically recommended for ELISA use.
• Matrix effects: If your samples are in a complex matrix (e.g., plasma, serum), ensure that the recombinant standard is diluted in a matrix-matched diluent to minimize matrix effects and improve accuracy.

Limitations and best practices:

• Not all recombinant proteins are suitable for all ELISAs: The recombinant β-NGF must be recognized by the capture and detection antibodies in your specific ELISA. Some recombinant proteins may lack certain post-translational modifications or conformations present in native β-NGF, potentially affecting antibody binding.
• Interference from related proteins: Be aware of potential cross-reactivity or interference from proNGF or other NGF isoforms, which can affect quantification in some ELISAs.
• Validation: If you are developing your own ELISA or using a non-kit-based assay, validate the recombinant standard for parallelism and recovery in your sample matrix.

Summary:
Recombinant human β-NGF is appropriate as a standard for ELISA quantification if it is validated for use in your assay system, matches the native protein in antibody recognition, and is prepared and diluted according to best practices for standard curve generation. Always consult your ELISA kit’s documentation or perform validation experiments if using a recombinant standard not supplied with the kit.

Recombinant Human β-NGF has been validated for a range of applications in published research, primarily in bioassays, cell culture studies, and as a standard in ELISA. Its biological activity has been demonstrated in both in vitro and in vivo models, with applications spanning neuroscience, regenerative medicine, and ophthalmology.

Key validated applications include:

• Bioassays:

  • Used to assess neuronal survival, differentiation, and neurite outgrowth in various cell types, including human, mouse, rat, and canine cells.
  • Demonstrated to induce proliferation of TF-1 human erythroleukemic cells in a dose-dependent manner.
  • Applied in studies modeling neurodegenerative diseases (e.g., ALS, Alzheimer’s), diabetic neuropathy, and traumatic brain injury.

• ELISA (Enzyme-Linked Immunosorbent Assay):

  • Used as a standard and capture reagent in ELISA protocols to quantify NGF levels in biological samples.

• Tissue Engineering and Regenerative Medicine:

  • Incorporated into bioengineered corneal stromal lenticules for sustained release, supporting corneal nerve regeneration and wound healing in vitro and in vivo.
  • Shown to accelerate wound healing and nerve regeneration in preclinical models, including ocular surface diseases and endochondral fracture repair.

• Ophthalmology and Retinal Research:

  • Evaluated for delivery to the retina and treatment of retinal degenerative diseases such as glaucoma, retinitis pigmentosa, and optic neuropathies.
  • Used in clinical and preclinical studies for corneal wound healing, improvement of corneal sensitivity, and nerve regeneration.

• Cancer Research:

  • Studied for its role in neuro-mesenchymal interactions and cancer progression, particularly in colorectal cancer models.

• Stem Cell and Neuronal Differentiation Studies:

  • Used to promote neural specification, differentiation, and migration in stem cell-derived models.

Additional validated uses:

• Drug Delivery Research:

  • Encapsulated in microparticles or microrods for localized and sustained delivery in tissue engineering applications.

• In Vivo Animal Models:

  • Applied in studies involving nerve injury, fracture repair, and neurodegeneration in rodents and other species.

Summary Table: Validated Applications of Recombinant Human β-NGF

These applications are supported by peer-reviewed studies and product validation data, confirming the utility of recombinant human β-NGF in diverse experimental systems.

To reconstitute and prepare Recombinant Human β-NGF protein for cell culture experiments, follow these best-practice steps:

1. Centrifuge the vial briefly (e.g., 3000 rpm for 5 minutes) before opening to ensure all lyophilized protein is at the bottom.

2. Reconstitution:

   • Use sterile water or sterile PBS (pH 7.2–7.4) as the solvent. The choice depends on the protein’s formulation and your downstream application. If the product was lyophilized from an acidic buffer, PBS is often preferred to quickly neutralize the pH.
   • Recommended concentration: Reconstitute to a stock concentration of 0.1–0.2 mg/mL (100–200 μg/mL). For example, add 1 mL of solvent to 0.1 mg (100 μg) of protein for a 0.1 mg/mL solution.
   • Carrier protein: For stability, especially for storage or low-concentration working solutions, add 0.1%–1% BSA or HSA (endotoxin-free) to the buffer. This prevents adsorption to plastic and loss of activity.
   • Mixing: Gently swirl or tap the vial to dissolve. Do not vortex or shake vigorously, as this can denature the protein.

3. Incubation: Allow the solution to sit at room temperature for at least 20 minutes to ensure complete dissolution.

4. Aliquoting and Storage:

   • Prepare small aliquots to avoid repeated freeze-thaw cycles.
   • Short-term storage (up to 1 week): 2–8 °C.
   • Long-term storage: −20 °C or −80 °C.
   • Always include carrier protein in storage buffer for stability.

5. Working solution preparation:

   • Dilute the stock solution to the desired final concentration in your cell culture medium immediately before use.
   • If possible, include a small amount of carrier protein in the working solution to maintain stability, especially at low concentrations.

Summary Table: β-NGF Reconstitution for Cell Culture

Additional Notes:

• Always consult the specific product’s Certificate of Analysis (CoA) or datasheet for any unique instructions.
• Avoid repeated freeze-thaw cycles to preserve protein activity.
• For sensitive applications, ensure all reagents are endotoxin-free.

These steps will help ensure maximum activity and stability of recombinant human β-NGF in your cell culture experiments.