Recombinant Human/Mouse NT-3

Recombinant Human/Mouse Neurotrophin-3 (NT-3) is a valuable tool for neuroscience and regenerative medicine research due to its well-characterized biological activities and broad applicability across experimental models. Here are key reasons to use Recombinant Human/Mouse NT-3 in your research:

1. Promotes Neuronal Survival, Growth, and Differentiation

NT-3 supports the survival, growth, and differentiation of neurons in both the peripheral and central nervous systems. It is particularly important for sensory neurons, motor neurons, and synapses, making it ideal for studies on neural development, regeneration, and neuroprotection.

2. Stimulates Axon Outgrowth and Regeneration

NT-3 enhances neurite outgrowth and axonal regeneration in vitro and in vivo. This is especially relevant for research on peripheral nerve injury, neuropathies (e.g., Charcot-Marie-Tooth disease), and spinal cord injury.

3. Modulates Synaptic Function and Plasticity

NT-3 modulates neurotransmitter release and synaptic plasticity, which is useful for studies on learning, memory, and neurological disorders such as Alzheimer’s disease.

4. Supports Schwann Cell Function

NT-3 is a key autocrine factor for Schwann cells, promoting their survival, differentiation, and myelination. This makes it essential for studies on peripheral nerve regeneration and demyelinating diseases.

5. Exhibits Anti-inflammatory and Neuroprotective Effects

NT-3 reduces oxidative stress, apoptosis, and inflammatory responses in neural cells. It suppresses pro-inflammatory cytokines (e.g., TNF-α, IL-1β) and enhances anti-inflammatory markers (e.g., IL-10, Foxp3), supporting its use in models of neuroinflammation and neurodegenerative diseases.

6. Activates TrkC and Other Tyrosine Kinase Receptors

NT-3 primarily activates the TrkC receptor, but can also interact with TrkA and TrkB in certain contexts, allowing for investigation of neurotrophin signaling pathways and receptor cross-talk.

7. Cross-Species Compatibility

Recombinant Human/Mouse NT-3 is biologically active in both human and mouse cell systems, enabling its use in a wide range of preclinical models, including transgenic mice and primary neuronal cultures.

8. Well-Validated in Disease Models

NT-3 has demonstrated efficacy in animal models of Alzheimer’s disease, peripheral neuropathy, spinal cord injury, and myocardial infarction, supporting its relevance for translational research.

9. Versatile Applications

NT-3 is suitable for:

• Neuronal cell culture and differentiation studies
• Functional assays (e.g., neurite outgrowth, survival)
• Investigation of neurotrophin signaling pathways
• In vivo studies (e.g., intramuscular, intracerebral, or systemic administration)
• Co-culture systems with Schwann cells or other glial cells

10. High Purity and Consistency

Commercially available recombinant NT-3 is produced under controlled conditions, ensuring high purity, batch-to-batch consistency, and reproducibility in experimental results.

In summary, Recombinant Human/Mouse NT-3 is a powerful and versatile reagent for studying neurotrophic mechanisms, neural regeneration, neuroprotection, and neuroinflammation, with broad relevance across basic and translational neuroscience research.

You can use recombinant human/mouse NT-3 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 and detection system used in your assay.

Key considerations and supporting details:

• Sequence Homology: Due to the high sequence homology between human and mouse NT-3, recombinant human NT-3 is commonly used as a standard for quantifying both human and rodent NT-3 in ELISA assays. This is a standard practice in commercial ELISA kits, which often use recombinant human NT-3 as the calibrator for both species.

• ELISA Kit Design: Many ELISA kits for NT-3 quantification are validated using recombinant NT-3 as the standard, and the assay is calibrated by generating a standard curve with serial dilutions of the recombinant protein. The concentration of NT-3 in unknown samples is then interpolated from this standard curve.

• Validation: It is essential to ensure that the recombinant NT-3 you use as a standard is compatible with your assay’s antibodies (i.e., the antibodies recognize the recombinant form equivalently to the native protein in your samples). Most commercial kits are validated for both natural and recombinant NT-3, but if you are developing your own assay, you should verify this equivalence.

• Species Cross-Reactivity: If your ELISA is designed for mouse NT-3 but uses recombinant human NT-3 as a standard, confirm that the antibodies used in the assay cross-react with both species. Most anti-NT-3 antibodies are raised against conserved regions and will detect both human and mouse NT-3, but this should be verified in your assay documentation or by preliminary testing.

• Standard Preparation: Prepare the standard curve by serially diluting the recombinant NT-3 in the same buffer or diluent used for your samples, following the protocol recommended for your specific ELISA. Ensure accurate pipetting and mixing to minimize variability.

• Documentation: Always refer to your ELISA kit’s manual or your assay protocol for specific instructions regarding standard preparation and compatibility. Do not mix standards or components from different manufacturers unless validated for cross-use.

Summary Table: Use of Recombinant NT-3 as ELISA Standard

Best Practices:

• Use recombinant NT-3 of known concentration and high purity.
• Validate that your assay’s antibodies detect the recombinant form equivalently to the native protein.
• Prepare the standard curve in the same matrix as your samples when possible.
• Follow the ELISA kit or assay protocol for standard preparation and curve fitting.

In summary, recombinant human/mouse NT-3 is widely accepted and routinely used as a standard for ELISA quantification and calibration, provided the above conditions are met.

Recombinant Human/Mouse NT-3 has been validated in published research for several key applications, primarily in the fields of neuroscience and cell biology. The most common validated applications include:

• Cell culture assays: NT-3 is widely used to support the growth, survival, and differentiation of neuronal cells in vitro, including primary neurons and neural stem/progenitor cells.
• Neuronal differentiation protocols: NT-3 is incorporated into protocols to induce or enhance neuronal differentiation, often in combination with other neurotrophic factors.
• Binding assays: Recombinant NT-3 is used to study receptor-ligand interactions, particularly with TrkC and related tyrosine kinase receptors.
• Functional bioactivity assays: NT-3 bioactivity is validated using luciferase reporter assays in HEK293T cells co-transfected with TrkA or TrkC receptors, measuring downstream signaling activation.
• Ex vivo and in vivo models of neural injury and repair: NT-3 has been used in models of myelin repair following ischemic damage, peripheral neuropathy, and neurodegenerative disease to assess its therapeutic potential.
• Therapeutic studies in animal models: Recombinant NT-3 has been administered in vivo (e.g., subcutaneous injection, gene therapy) to evaluate effects on axonal regeneration, myelination, and functional recovery in models of Charcot–Marie–Tooth disease and Alzheimer's disease.

Supporting details:

• In cell culture, NT-3 is validated for promoting neuronal survival and differentiation, often measured by morphological changes, marker expression, or functional assays.
• In binding assays, NT-3 is used to characterize its interaction with TrkC and other neurotrophin receptors, supporting studies of receptor specificity and downstream signaling.
• In functional bioactivity assays, NT-3's ability to activate TrkA/TrkC is quantified by luciferase reporter systems, confirming its biological activity.
• In ex vivo models, NT-3 has been applied to whole cells or tissue explants to study myelin repair and neuroprotection after injury.
• In vivo, recombinant NT-3 has demonstrated efficacy in improving nerve regeneration, myelination, and functional outcomes in rodent models of neuropathy and neurodegeneration.

Additional relevant information:

• NT-3 is also used in research on microglial activation, synaptic plasticity, and neuroprotection against amyloid-β toxicity.
• The protein is validated for use in both human and mouse systems, with identical mature sequences, facilitating cross-species studies.
• NT-3's applications are strictly for research use and not for diagnostic or therapeutic purposes in humans or animals.

In summary, Recombinant Human/Mouse NT-3 is validated for cell culture, neuronal differentiation, receptor binding, bioactivity assays, and therapeutic studies in published research, with a strong focus on neural growth, survival, differentiation, and repair.

To reconstitute and prepare Recombinant Human/Mouse NT-3 protein for cell culture experiments, dissolve the lyophilized protein in sterile water or buffer to a concentration of ≥100 µg/mL, avoiding vortexing or vigorous pipetting. For optimal stability and activity, further dilute in a buffer containing a carrier protein such as 0.1% BSA or serum albumin.

Step-by-step protocol:

• Centrifuge the vial briefly before opening to ensure all lyophilized material is at the bottom.
• Add sterile water or buffer (e.g., 10 mM HCl, PBS, or ddH₂O) to achieve a concentration of at least 100 µg/mL.
• Gently mix by swirling or inverting; do not vortex or pipette vigorously to avoid protein denaturation.
• If required for your application, add carrier protein (e.g., 0.1% BSA or serum albumin) to prevent adsorption and loss of activity during storage and handling.
• Aliquot the solution into single-use portions to minimize freeze-thaw cycles.
• Storage:
  • Short-term: Store reconstituted protein at 2–8°C for up to 1 week.
  • Long-term: Store aliquots at –20°C to –80°C for several months.
  • Avoid repeated freeze-thaw cycles to preserve bioactivity.

Preparation for cell culture:

• Before use, dilute the NT-3 stock solution into your cell culture medium to the desired final concentration, ensuring compatibility with your assay conditions.
• NT-3 is bioactive as a non-covalently linked homodimer and is typically used in neuronal survival, differentiation, or signaling assays.

Additional notes:

• The mature NT-3 sequence is identical in human, mouse, and rat, so the same protocol applies for all three species.
• Always consult the specific product datasheet for buffer recommendations and concentration limits, as formulations may vary between suppliers.

Summary of key points:

• Reconstitute at ≥100 µg/mL in sterile water, PBS, or 10 mM HCl.
• Do not vortex; mix gently.
• Add carrier protein for stability if required.
• Aliquot and store at –20°C to –80°C for long-term use.
• Dilute into cell culture medium immediately before use.

This protocol ensures maximal stability and bioactivity of NT-3 for cell culture experiments.