Recombinant Mouse Beta-nerve growth factor (Ngf) (Active)

What is the molecular structure of recombinant mouse β-NGF?

Recombinant mouse β-NGF functions as a non-covalently linked homodimer composed of two 13.4-13.5 kDa polypeptide monomers. Each monomer contains 120 amino acids with three disulfide bonds that are essential for biological activity . The active region of the protein typically spans from Ser122 to Gly241 of the full-length precursor protein . The functional homodimeric structure adopts a characteristic configuration belonging to the cysteine-knot family of growth factors, which is critical for receptor binding and signaling pathway activation .

What signaling pathways does mouse β-NGF activate in target cells?

Mouse β-NGF initiates signaling through two primary receptors: the low-affinity nerve growth factor receptor (LNGFR) and the high-affinity tropomyosin receptor kinase A (TrkA) . Upon binding to these receptors, β-NGF activates multiple downstream signaling cascades including:

• Phosphatidylinositol 3-kinase (PI3K) pathway - promoting cell survival

• Ras-MAPK pathway - regulating cell differentiation and growth

• Phospholipase C (PLC) signaling - modulating calcium homeostasis and further downstream effects

These pathways collectively regulate neuronal survival, axonal growth, synaptic plasticity, and cellular differentiation in target tissues . Cross-linking studies with labeled NGF have demonstrated that the high-affinity receptor forms a approximately 158 kDa complex with NGF, while the low-affinity receptor forms a 100 kDa complex .

How do mouse, rat and human β-NGF compare in terms of homology and cross-reactivity?

Comparative analysis of β-NGF across species reveals significant homology:

What are the validated cell-based assays for measuring recombinant mouse β-NGF activity?

Several established bioassays can reliably measure the biological activity of recombinant mouse β-NGF:

• PC12 Cell Differentiation Assay: PC12 cells (rat pheochromocytoma) undergo neuronal differentiation with neurite outgrowth when exposed to biologically active β-NGF. This morphological change can be quantified by measuring total neurite length per cell and counting cells bearing at least one axon longer than the cell body .

• TF-1 Cell Proliferation Assay: Human erythroleukemic TF-1 cells proliferate in response to β-NGF in a dose-dependent manner. The effective dose for 50% response (ED50) typically ranges from 0.3-1.5 ng/mL . This assay can be quantified using metabolic indicators like Rezazurin .

• Sympathetic Ganglion Bioassay: This classical assay measures neuronal survival and neurite outgrowth from sympathetic ganglia exposed to β-NGF. Both human and mouse NGF demonstrate similar activity profiles in this system .

• Neutralization Assays: Anti-β-NGF antibodies can be used to confirm specificity, with neutralization typically achieved at concentrations of 0.05-0.5 μg/mL in the presence of 5 ng/mL recombinant mouse β-NGF .

How should recombinant mouse β-NGF be reconstituted and stored for optimal stability?

For maintaining maximum biological activity of recombinant mouse β-NGF, follow these research-validated protocols:

Reconstitution should be performed using a sterile buffer such as 20mM Tris with 150mM NaCl, pH 8.0, or as specified in the product documentation . To prevent protein adsorption to container surfaces, addition of carrier proteins (e.g., 0.1% BSA) may be beneficial for dilute solutions. Importantly, repeated freeze-thaw cycles significantly reduce biological activity and should be strictly avoided .

What factors affect the binding of recombinant mouse β-NGF to laboratory materials and how can non-specific binding be minimized?

Non-specific binding of β-NGF to laboratory materials, particularly plastics and delivery systems, presents a significant challenge for accurate dosing in experiments. Research has identified several key factors:

• Material Composition: β-NGF exhibits varying degrees of adsorption to different laboratory plastics, glass, and metals in delivery systems such as infusion pumps and catheters .

• Protein Concentration: Lower concentrations of β-NGF are more susceptible to significant loss through non-specific binding.

• Buffer Composition: Ionic strength, pH, and the presence of carrier proteins significantly impact adsorption rates.

To minimize non-specific binding, researchers should implement these evidence-based strategies:

• Use blocking formulations containing carrier proteins such as bovine serum albumin (0.1-1%)

• Pre-treat surfaces with Pluronic F-127 or similar surfactants

• Use siliconized or low-protein-binding plasticware

• Prepare stock solutions at higher concentrations

• Include 0.05% Tween-20 in storage buffers when compatible with experimental design

How can researchers accurately quantify recombinant mouse β-NGF in experimental samples?

Accurate quantification of β-NGF in experimental samples requires validated analytical techniques:

• Two-site Enzyme Immunoassay (EIA): This high-sensitivity approach can detect β-NGF at concentrations as low as 1 pg/mL. Monoclonal antibodies like 27/21 have been optimized for both mouse and human NGF detection, making this method suitable for cross-species studies .

• Western Blotting: For semi-quantitative analysis, Western blotting using specific anti-β-NGF antibodies can confirm protein presence and approximate quantity.

• Functional Bioassays: As described in section 2.1, cell-based assays provide a measure of biological activity rather than absolute protein concentration. The TF-1 proliferation assay demonstrates a dose-dependent response that can be used to establish a standard curve for unknown samples .

• Mass Spectrometry: For advanced research applications requiring precise identification and quantification, LC-MS/MS techniques can be employed, especially when analyzing complex biological samples.

How does recombinant mouse β-NGF therapy impact bone marrow sensory innervation in diabetic models?

Research investigating NGF gene therapy in diabetic mouse models has revealed significant impacts on bone marrow sensory innervation and stem cell mobilization:

• Neuropathy Reversal: Type 1 diabetic mice exhibit sensory neuropathy in bone marrow characterized by reduced substance P expression. Administration of NGF gene therapy significantly increased substance P-positive nerve fibers in bone marrow (approximately 2-fold increase compared to untreated diabetic controls) .

• Cellular Activation: NGF treatment increased the number of cells co-expressing substance P and phosphorylated ribosomal protein S6 (P-rpS6) in bone marrow, indicating enhanced cellular activity and metabolic function .

• Stem Cell Mobilization: Following peripheral ischemia, NGF-treated diabetic mice demonstrated improved mobilization of substance P-responsive stem cells from bone marrow compared to untreated diabetic controls .

• Healing Response: The enhanced sensory innervation resulting from NGF therapy translated to improved healing responses after peripheral ischemic events, suggesting therapeutic potential for diabetic complications .

These findings establish bone marrow nociceptors as potential therapeutic targets for addressing ischemic complications in diabetes and highlight the importance of sensory innervation in regulating stem cell function and tissue repair mechanisms .

What are the considerations for using recombinant mouse β-NGF in Alzheimer's disease research models?

Alzheimer's disease research using recombinant mouse β-NGF requires careful consideration of several factors:

• Cholinergic Neuron Targeting: NGF has demonstrated ability to rescue cholinergic neurons both in vitro and in vivo, addressing one of the early changes in Alzheimer's disease—loss of cholinergic function .

• Delivery Challenges: Direct administration to the central nervous system via intracerebroventricular (ICV) routes requires specialized infusion pumps and catheters with controlled release properties. Researchers must validate delivery systems to ensure consistent dosing given NGF's binding properties .

• Cross-species Considerations: When using mouse models to study human disease, researchers should note the approximately 90% homology between mouse and human β-NGF. For translational studies, human recombinant NGF may be more appropriate, though mouse NGF remains valuable for proof-of-concept studies .

• Dosage Optimization: Effective doses for neuroprotection must be carefully titrated, as excessive NGF can cause unwanted effects including pain hypersensitivity and aberrant neuronal sprouting.

• Combined Therapeutic Approaches: Research suggests that combining NGF therapy with other treatments targeting different Alzheimer's disease mechanisms may provide synergistic benefits.

How does the biological activity of bacterially-expressed recombinant mouse β-NGF compare with mammalian cell-derived protein?

The expression system significantly impacts the properties of recombinant mouse β-NGF:

E. coli-expressed recombinant mouse β-NGF (such as the protein described in result ) maintains high biological activity despite lacking post-translational modifications, with typical ED50 values of 0.3-1.5 ng/mL in TF-1 cell proliferation assays . This indicates that the primary structure and correct disulfide bond formation—rather than glycosylation—are the critical determinants of β-NGF activity.

What are the validated methods for evaluating neuronal differentiation in response to recombinant mouse β-NGF treatment?

Researchers can employ several established methods to quantitatively assess neuronal differentiation induced by β-NGF:

• Morphological Analysis:

  • Neurite outgrowth measurement: Total neurite length per cell

  • Branching complexity: Number of branch points per neurite

  • Neuron-like cell counting: Cells bearing at least one axon longer than the cell body

• Molecular Marker Analysis:

  • Upregulation of neuron-specific proteins (β-III-tubulin, MAP2, NeuN)

  • Expression of synaptic markers (synaptophysin, PSD-95)

  • Activation of TrkA receptor and downstream signaling components

• Functional Assessments:

  • Electrophysiological recording of action potentials

  • Calcium imaging to detect neuronal activity

  • Neurotransmitter release assays

In PC12 cell studies, NGF-conditioned medium significantly increased total neurite length per cell (p<0.0001 vs. control) and the number of neuron-like cells (p<0.01 vs. control) under both normal and high-glucose conditions, demonstrating the robust neuronal differentiation capacity of biologically active β-NGF even under diabetic-like conditions .