Recombinant Human NT-3

Recombinant Human NT-3 (Neurotrophin-3) is widely used in research applications to promote the survival, differentiation, and growth of neurons and glial cells, making it essential for studies in neuroscience, stem cell biology, and regenerative medicine.

NT-3 is a member of the neurotrophin family and primarily activates the TrkC tyrosine kinase receptor, but can also interact with TrkA and TrkB in certain contexts. Its biological functions include:

• Neuronal survival and differentiation: NT-3 supports the survival and differentiation of existing neurons and glial cells, and stimulates the growth of new nerve cells.
• Neurite outgrowth and synaptic modulation: NT-3 induces neurite outgrowth and modulates neurotransmitter release at synapses, which is critical for neural network formation and plasticity.
• Stem cell applications: NT-3 is used to maintain pluripotency and direct differentiation of embryonic stem cells (ESCs), induced pluripotent stem cells (iPSCs), and neural progenitors into neuronal and glial lineages.
• Disease modeling and neuroprotection: NT-3 has been shown to reduce neuronal loss in disease models (e.g., experimental meningitis), and may play a role in treating aspects of neuropathic pain and neurodegenerative conditions.
• Developmental studies: NT-3 is involved in embryonic neuronal development and maintenance of the adult nervous system.

Experimental advantages of recombinant NT-3:

• High purity and low endotoxin levels: Recombinant preparations are typically animal-component and carrier-protein free, minimizing variability and risk of contamination in sensitive cell culture and in vivo studies.
• Species cross-reactivity: The mature NT-3 peptide is highly conserved across mammals, allowing use in human, mouse, rat, and porcine models.
• Defined activity: Recombinant NT-3 provides consistent and reproducible biological activity, essential for quantitative assays and mechanistic studies.

Typical applications include:

• Cell culture assays for neuronal and glial survival/differentiation.
• Stem cell differentiation protocols.
• Neuroscience research, including synaptic function and neuroprotection.
• In vivo studies of neural development and injury response.

In summary, using recombinant human NT-3 enables precise control over neurotrophic signaling in experimental systems, facilitating research into neural development, disease mechanisms, and regenerative therapies.

Yes, recombinant human NT-3 is well-suited for use as a standard for quantification and calibration in ELISA assays. This is a standard practice in NT-3 detection protocols.

Recommended Use and Formulation

Recombinant human NT-3 produced through baculovirus expression systems (such as Sf21-derived protein) is commonly employed as the calibration standard in sandwich ELISA formats. The protein is typically supplied in a lyophilized form and should be reconstituted at appropriate concentrations in sterile phosphate-buffered saline (PBS) containing carrier proteins such as bovine serum albumin (BSA). A reconstitution concentration of 50 μg/mL in PBS with at least 0.1% serum albumin is standard practice.

Assay Performance Characteristics

When using recombinant NT-3 as your standard, you can expect the following performance metrics:

Detection Range and Sensitivity: Standard curves typically span from approximately 4-3000 pg/mL, with sensitivities reaching as low as 4 pg/mL depending on the assay configuration. Some protocols achieve detection ranges extending to 10 ng/mL with sensitivities below 0.055 ng/mL.

Precision: Inter-assay coefficient of variation is typically less than 12%, while intra-assay variation remains below 10%.

Specificity: Recombinant NT-3 standards demonstrate excellent specificity with no significant cross-reactivity with structural analogues or other neurotrophic factors.

Important Considerations

When preparing your standard curve, ensure that the recombinant protein is diluted using the sample diluent buffer provided with your specific assay kit rather than alternative buffers. Additionally, avoid repeated freeze-thaw cycles by using a manual defrost freezer for storage, as this preserves protein activity and ensures consistent calibration across experiments.

The recombinant standard should be measured at 450 nm ± 10 nm following substrate development, and results are determined by comparing sample optical density values against the standard curve through regression analysis.

Recombinant Human NT-3 has been validated for several key applications in published research, primarily in the fields of neuroscience, cell biology, and regenerative medicine.

Validated Applications:

• Bioassays: NT-3 is widely used in bioassays to assess its neurotrophic activity, including promotion of neuronal survival, differentiation, and neurite outgrowth in various cell types such as PC12 cells, human pluripotent stem cells, and primary neurons.
• Cell Culture: NT-3 is routinely applied as a supplement in neuronal and glial cell culture systems to support cell survival, proliferation, and differentiation, especially for sensory and motor neurons.
• Stem Cell Differentiation: NT-3 is used in protocols to induce differentiation of pluripotent stem cells or neural crest stem cells into functional neurons, including β-tubulin III+ peripheral neurons.
• Neurite Outgrowth Assays: NT-3 has been validated for stimulating neurite extension in neuronal cell lines and primary neurons, often measured by microscopy and molecular markers such as GAP43 signaling.
• In Vivo Studies: NT-3 has been administered in animal models to study its effects on nerve regeneration, axonal growth, myelination, and functional recovery after injury, including models of neuropathy and neurodegenerative disease.
• Functional Assays: NT-3 is used to investigate TrkC receptor signaling, ERK1/2 MAPK pathway activation, and other downstream effects in neuronal cells.
• Proangiogenic Assays: NT-3 has demonstrated proangiogenic effects in endothelial cell survival and angiogenesis studies.
• Organoid Models: NT-3 has been applied to human organoid systems to study neurodevelopment and disease mechanisms.

Representative Published Research:

• Neuronal Differentiation: NT-3 promotes differentiation of human pluripotent stem cells into functional neurons and supports long-term survival in rodent hippocampus models.
• Neuroregeneration: NT-3 enhances axonal regeneration and myelination in peripheral nerve injury and neuropathy models, with demonstrated improvements in motor function and histopathology.
• Neuroprotection: NT-3 reduces neuronal loss in models of meningitis and mitigates oxidative stress in neurodegenerative disease models.
• Cellular Mechanisms: NT-3 induces ERK1/2 MAPK phosphorylation and activates TrkC signaling in neuronal cells, supporting its role in cell survival and differentiation.

Summary Table of Validated Applications

These applications are supported by multiple peer-reviewed studies and product validation data, confirming the utility of recombinant human NT-3 in both basic and translational neuroscience research.

To reconstitute and prepare Recombinant Human NT-3 protein for cell culture experiments, follow these steps for optimal protein stability and biological activity:

1. Reconstitution

• Centrifuge the vial briefly before opening to ensure all lyophilized material is at the bottom.
• Add sterile water or PBS: Most protocols recommend reconstituting NT-3 in sterile distilled water or sterile PBS. The typical concentration is 0.1–1.0 mg/mL for stock solutions. Some protocols specify 50–100 μg/mL as a working concentration.
• Carrier protein: For improved stability, especially for storage or dilute solutions, add 0.1% BSA (bovine serum albumin) or HSA (human serum albumin) to the buffer.
• Mix gently: Do not vortex or pipette vigorously. Allow the protein to dissolve by gentle swirling or by letting it sit at room temperature for 15–30 minutes. If undissolved material remains, gently mix for up to 2 hours at room temperature.

2. Aliquoting and Storage

• Aliquot the reconstituted protein into small volumes to avoid repeated freeze-thaw cycles.
• Short-term storage: Store at 2–8°C for up to 1 week.
• Long-term storage: For extended storage, freeze aliquots at –20°C to –80°C.
• Carrier protein is recommended for long-term storage to prevent adsorption and loss of activity.

3. Preparation for Cell Culture

• Dilute the stock solution to the desired working concentration using cell culture medium or buffer containing 0.1% BSA/HSA to maintain stability.
• Sterility: Ensure all solutions and diluents are sterile to prevent contamination.
• Avoid repeated freeze-thaw cycles as this can reduce protein activity.

Summary Table: Key Steps for NT-3 Reconstitution

Additional Notes

• Always consult the specific product datasheet for any unique instructions.
• If using for sensitive cell types, confirm that the carrier protein (BSA/HSA) is compatible with your assay.

These steps will help ensure maximum stability and bioactivity of recombinant NT-3 in your cell culture experiments.