Recombinant Human NT-4

Recombinant Human NT-4 (Neurotrophin-4) is used in research applications to study and manipulate neuronal survival, differentiation, and growth, as well as to investigate neurotrophic signaling pathways in both central and peripheral nervous system models. It is also increasingly applied in developmental biology and regenerative medicine contexts.

Key reasons to use Recombinant Human NT-4 in research include:

• Neuronal Survival and Differentiation: NT-4 is a member of the neurotrophin family, which are essential growth factors for the development, maintenance, and survival of neurons. NT-4 specifically promotes the survival and differentiation of selected populations of peripheral and central nervous system neurons, often via activation of the TrkB receptor.

• Neurite Outgrowth and Synaptic Plasticity: NT-4 supports neurite extension, dendritic outgrowth, and synaptic connectivity, making it valuable for studies on neuronal network formation and plasticity.

• Cell Signaling Studies: NT-4 activates key intracellular signaling pathways, such as the MAPK/ERK pathway, which are involved in cell survival, proliferation, and differentiation. This makes it a useful tool for dissecting neurotrophic signaling mechanisms.

• Developmental Biology: NT-4 has been shown to influence early embryonic development, including the specification of trophectoderm lineage and regulation of apoptosis-related genes in blastocyst models, indicating its broader role beyond the nervous system.

• Disease and Injury Models: NT-4 is used to model neurodegenerative diseases, nerve injury, and regeneration, as it can protect against apoptotic neuronal death and promote recovery of neural function.

• Defined, Reproducible Reagent: Recombinant NT-4 provides a highly pure, biologically active, and consistent protein source, which is critical for reproducibility in cell culture, biochemical assays, and animal studies.

Additional context:

• NT-4 shares high homology with rodent forms, allowing for cross-species studies.
• It is often used alongside other neurotrophins (e.g., BDNF, NGF) to compare or synergize effects in neuronal cultures and developmental models.
• Recombinant NT-4 is suitable for in vitro and in vivo applications, including cell-based assays, organotypic cultures, and animal models.

In summary, Recombinant Human NT-4 is a versatile tool for investigating neurobiology, developmental processes, and regenerative strategies due to its well-characterized, potent neurotrophic activities and defined molecular properties.

Yes, recombinant human NT-4 can be used as a standard for quantification or calibration in ELISA assays, provided it is of high purity and its concentration is accurately determined. This is a common practice in quantitative ELISA protocols for measuring NT-4 levels in biological samples.

Key considerations and best practices:

• Formulation: For ELISA standard preparation, it is generally recommended to use the recombinant NT-4 protein formulated with BSA (bovine serum albumin) as a carrier. BSA enhances protein stability and allows for more accurate and reproducible standard curves. Carrier-free formulations are available but are typically reserved for applications where BSA may interfere with detection.

• Purity and Quantification: The recombinant NT-4 should be highly purified, and its concentration should be verified using reliable methods such as HPLC or spectrophotometry. This ensures the accuracy of your standard curve and subsequent quantification.

• Standard Curve Preparation: Prepare a serial dilution of the recombinant NT-4 standard in the same buffer or matrix as your samples. The standard curve should cover the expected range of NT-4 concentrations in your samples, typically from low picogram to nanogram per milliliter levels.

• Validation: It is essential to validate that the recombinant NT-4 behaves similarly to endogenous NT-4 in your assay system. This includes confirming parallelism between the standard curve and sample dilution curves, and ensuring no matrix effects interfere with quantification.

• Data Analysis: Use appropriate curve fitting methods, such as four-parameter logistic (4PL) or five-parameter logistic (5PL) models, for standard curve analysis in ELISA.

Limitations and additional notes:

• The recombinant NT-4 standard should match the species and isoform of NT-4 you intend to measure. Using a standard from a different species or isoform may result in inaccurate quantification due to differences in antibody recognition.
• Some vendors note that ELISA standard recombinant proteins are not recommended for bioassay usage unless specifically validated for that purpose. However, for quantification and calibration in ELISA, recombinant NT-4 is suitable if it meets purity and concentration requirements.

Summary of protocol steps:

1. Reconstitute recombinant NT-4 according to manufacturer instructions, typically in PBS with 0.1% BSA.
2. Prepare serial dilutions to generate a standard curve.
3. Run ELISA with standards and samples in parallel.
4. Analyze data using appropriate curve fitting software.

In conclusion, recombinant human NT-4 is appropriate for use as a standard in ELISA quantification, provided it is properly formulated, quantified, and validated for your assay system.

Recombinant Human NT-4 (Neurotrophin-4) has been validated in published research for applications including neuronal survival assays, activation of intracellular signaling pathways, neuronal differentiation, dendritic outgrowth, and as a component in cancer-targeted drug delivery systems.

Key validated applications in published research:

• Neuronal survival and viability assays: NT-4 has been used to enhance cell survival in TrkB-expressing cell lines (e.g., 293-TrkB cells) under serum-free conditions, with cell viability assessed by MTS assay. It acts as a survival factor for specific populations of sensory neurons.

• Activation of signaling pathways: Recombinant NT-4 induces activation of ERK1/2 MAPK in TrkB-transfected HEK-293 cells, as demonstrated by phospho-ERK1/2 immunoblotting.

• Neuronal differentiation and dendritic outgrowth: NT-4 promotes dendritic outgrowth and calcium currents in cultured mesencephalic dopamine neurons, and supports growth and remodeling of adult motor neuron innervation.

• Sensitization to cell death: NT-4 has been shown to sensitize cortical neurons to cell death, indicating its role in neuronal stress response studies.

• Cancer-targeted drug delivery: NT4-based peptides have been developed as selective tumor-targeting agents for drug delivery, such as NT4-QD-PTX nanoparticles for early tumor detection and combined therapy/diagnosis in cancer models.

Additional validated uses include:

• Bioactivity assays: NT-4 is routinely used to confirm TrkB receptor activation and downstream signaling in various cell-based assays.
• Neuroprotection studies: NT-4 has been shown to protect against apoptotic neuronal death in vitro.

These applications are supported by peer-reviewed publications and product validation data, demonstrating NT-4’s utility in neurobiology, cell signaling, and targeted therapy research.

To reconstitute and prepare Recombinant Human NT-4 protein for cell culture experiments, follow these best-practice steps:

1. Centrifuge the vial
   Before opening, briefly centrifuge the lyophilized protein vial (10,000 × g for 20–30 seconds) to ensure all material is at the bottom and not lost when opening.

2. Reconstitution buffer

   • For most cell culture applications, reconstitute NT-4 in sterile PBS (phosphate-buffered saline) or sterile deionized water.
   • To enhance protein stability and prevent adsorption to plastic, add 0.1%–0.5% carrier protein such as bovine serum albumin (BSA) or human serum albumin.

3. Concentration

   • Common reconstitution concentrations are 50 μg/mL in PBS with 0.1% BSA, or 100 μg/mL in sterile water.
   • For more dilute working solutions, maintain at least 0.1 mg/mL protein or supplement with BSA to prevent loss by adsorption.

4. Mixing

   • Gently mix by pipetting or swirling. Do not vortex, as this may denature the protein.

5. Aliquot and storage

   • After reconstitution, aliquot the solution to avoid repeated freeze-thaw cycles.
   • Store aliquots at –20°C to –80°C for long-term storage, or at 4°C for short-term use (up to 1 month) in the presence of carrier protein.
   • Avoid multiple freeze-thaw cycles, as this can reduce activity.

6. Preparation for cell culture

   • Before use, dilute the reconstituted NT-4 to the desired working concentration in cell culture medium, ensuring the presence of carrier protein if further dilution is required.
   • Typical working concentrations for cell-based assays range from 0.1 to 100 ng/mL, depending on the cell type and experimental design.

Summary protocol example:

• Centrifuge vial (10,000 × g, 20–30 s).
• Add sterile PBS + 0.1% BSA to achieve 50 μg/mL.
• Gently mix to dissolve (do not vortex).
• Aliquot and store at –20°C or –80°C.
• Dilute to working concentration in cell culture medium with carrier protein.

Additional notes:

• Always consult the specific product’s Certificate of Analysis or datasheet for any manufacturer-specific recommendations, as formulations may vary.
• If using for sensitive or serum-free cultures, ensure all reagents are endotoxin-free and sterile.

This protocol ensures maximal stability and biological activity of recombinant NT-4 for cell culture applications.