Recombinant Mouse β-NGF

Recombinant Mouse β-NGF is widely used in research because it is a well-characterized neurotrophin essential for the survival, differentiation, and maintenance of sympathetic and sensory neurons, and it plays a key role in neurogenesis, neuroprotection, and tissue repair.

Key reasons to use recombinant Mouse β-NGF in research applications:

• Neuronal Survival and Differentiation: β-NGF is critical for promoting the survival and differentiation of sympathetic and sensory neurons in the peripheral nervous system, and it also acts as a trophic factor for basal forebrain cholinergic neurons in the central nervous system.
• Neuroprotection and Regeneration: It is a potent neuroprotective agent, preventing neuronal degeneration and supporting neuron regeneration in various injury and disease models.
• Tissue Repair and Healing: Recombinant β-NGF accelerates endochondral fracture repair, enhances bone healing, and promotes functional restoration after nerve injury. It has also been shown to facilitate healing in corneal and cutaneous ulcers.
• Defined, Reproducible Activity: Recombinant proteins provide consistent, batch-to-batch reproducibility and eliminate variability associated with native or animal-derived NGF, which is crucial for controlled experimental design and reliable results.
• Versatility in Experimental Models: β-NGF is used in a wide range of applications, including in vitro bioassays, neuronal culture systems, animal models of neurodegeneration, tissue engineering, and regenerative medicine studies.
• Mechanistic Studies: It is essential for dissecting NGF-TrkA and p75NTR signaling pathways, which are involved in neuronal growth, survival, and synaptic plasticity.

In summary, using recombinant Mouse β-NGF allows for precise, reproducible investigation of neurotrophic mechanisms, neuroprotection, tissue repair, and regeneration in both basic and translational neuroscience research.

You can use recombinant mouse 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 determined, and it is compatible with the antibodies and detection system used in your assay.

Key considerations and supporting details:

• Purity and Form: The recombinant β-NGF should be highly purified and, ideally, in a form that closely mimics the native protein, as antibody recognition can be conformation-dependent. Carrier-free preparations are preferred for standard curve generation to avoid interference from stabilizing proteins such as BSA, unless your assay buffer contains similar components.

• Calibration and Quantification: ELISA kits often use either recombinant or native β-NGF as the standard. Recombinant standards are widely accepted for quantification, as long as the antibodies in your ELISA recognize both the recombinant and native forms equivalently. Many commercial ELISA kits are validated using recombinant β-NGF, and their standard curves are generated with this material.

• Parallelism: It is important to confirm that the dose-response curve generated with the recombinant standard is parallel to that of the endogenous β-NGF in your sample matrix. This ensures that quantification is accurate and not affected by matrix effects or differences between recombinant and native protein.

• Protein Form Specificity: If your ELISA is designed to detect mature β-NGF, ensure your recombinant standard is also the mature form, not proNGF, as cross-reactivity between these forms is typically very low in well-validated assays. Using the wrong form as a standard can lead to inaccurate quantification.

• Documentation and Validation: Refer to the product datasheet or technical documentation for your recombinant β-NGF to confirm its suitability as an ELISA standard. Some suppliers specifically recommend their recombinant proteins for use as ELISA standards, while others may note that their proteins are not validated for this purpose.

Best Practices:

• Prepare a standard curve using serial dilutions of the recombinant β-NGF in the same buffer as your samples.
• Validate the standard curve by checking for parallelism with endogenous β-NGF in representative sample matrices.
• Use only for research purposes, not for diagnostic or clinical applications, unless specifically validated for such use.

In summary, recombinant mouse β-NGF is suitable as an ELISA standard if it is pure, well-characterized, and matches the form detected by your assay. Always confirm compatibility with your specific ELISA system and validate the standard curve for accurate quantification.

Recombinant Mouse β-NGF has been validated for a range of applications in published research, primarily focusing on its neurotrophic and bioactive properties. The most commonly validated applications include:

• Functional/Bioactivity Assays:
  Recombinant mouse β-NGF is widely used to assess its ability to promote survival, differentiation, and neurite outgrowth in neuronal cell cultures, such as PC12 cells and primary sympathetic neurons. These assays confirm its biological activity via TrkA and p75 receptor signaling.

• Cell-Based Assays:
  Used to stimulate or modulate cellular responses in various cell types, including neuronal, glial, and non-neuronal cells. For example, it has been used to induce proliferation in TF-1 human erythroleukemic cells and to study signaling pathways in mouse and rat neurons.

• ELISA (Enzyme-Linked Immunosorbent Assay):
  Validated for use as a standard or control in ELISA to quantify NGF levels in biological samples.

• Western Blot:
  Used as a positive control or to validate antibody specificity in Western blotting for NGF detection.

• Immunohistochemistry:
  Applied to tissue sections to validate NGF localization and expression patterns.

• In Vivo Studies:
  Recombinant mouse β-NGF has been used in animal models to study its effects on neuronal survival, regeneration, pain processing, and reproductive physiology. For example, it has been used to investigate ovulation mechanisms via GnRH neuron activation in mice, and to assess its role in wound healing and neuropathy models.

• Reproductive Biology:
  Used to study the impact of NGF on oocyte competence, cumulus cell communication, and ovulation in mouse models.

Supporting details and representative studies:

• Neuronal survival and differentiation: Extensively validated in PC12 cell assays and primary sympathetic neuron cultures, confirming its role as a potent neurotrophic factor.
• Pain and neuropathy models: Used in studies of small fiber neuropathy and pain signaling pathways in rodents.
• Reproductive studies: Applied to mouse oocyte and cumulus cell complexes to study NGF’s effects on fertility and ovulation.
• Wound healing: Recombinant murine NGF has demonstrated efficacy in accelerating wound healing in preclinical mouse models.

Summary Table of Validated Applications

These applications are well-supported by published research and are considered standard for validating the activity and specificity of recombinant mouse β-NGF in both in vitro and in vivo experimental systems.

To reconstitute and prepare Recombinant Mouse β-NGF protein for cell culture experiments, dissolve the lyophilized protein in sterile water or PBS to a concentration of at least 100 μg/mL, incubate at room temperature for 10–20 minutes, and gently mix to ensure complete dissolution. Avoid repeated freeze-thaw cycles and, if possible, include a carrier protein such as 0.1% BSA to enhance stability.

Detailed protocol:

• Centrifuge the vial briefly before opening to ensure all powder is at the bottom.
• Add sterile water or PBS: Most protocols recommend reconstituting in sterile water or PBS to a final concentration of 100 μg/mL or higher. For example, add 100 μL of sterile water to 100 μg of protein for a 1 mg/mL stock, or 1 mL for a 100 μg/mL stock.
• Carrier protein (optional but recommended): For long-term stability and to prevent adsorption to plastic, add at least 0.1% BSA (bovine serum albumin) to the reconstitution buffer if not already present.
• Mix gently: Do not vortex. Gently pipette up and down or swirl to dissolve the protein completely.
• Incubate at room temperature for 10–20 minutes to ensure full reconstitution.
• Aliquot and store: After reconstitution, aliquot the solution to avoid repeated freeze-thaw cycles. Store at 2–8 °C for up to 1 week, or at –20 °C to –80 °C for longer-term storage.
• Working dilution: Before adding to cell culture, dilute the stock solution to the desired working concentration using cell culture medium or buffer containing 0.1% BSA or other carrier protein to minimize loss from adsorption.

Key precautions:

• Avoid vigorous mixing or vortexing, which can denature the protein.
• Use sterile technique throughout to prevent contamination.
• Check the product datasheet for any protein-specific recommendations, as some preparations may require PBS or a specific pH for optimal solubility.

Summary table:

This protocol ensures maximum solubility, stability, and bioactivity of recombinant mouse β-NGF for cell culture applications.