Recombinant Human Neuroserpin

Recombinant Human Neuroserpin is used in research applications to study its roles as a serine protease inhibitor in the nervous system, particularly its regulation of tissue-type plasminogen activator (tPA), neuroprotection, synaptic plasticity, and involvement in neurological diseases.

Key scientific reasons to use recombinant human neuroserpin include:

• Neuroprotection: Neuroserpin administration has been shown to reduce cerebral infarct volumes and increase neuronal survival in models of ischemia and excitotoxicity, making it valuable for studying neuroprotective mechanisms and potential therapeutic strategies for stroke and neurodegeneration.

• Synaptic Plasticity and Axogenesis: Neuroserpin regulates axonal growth and synapse formation. It enhances dendritic spine density and morphology, correlating with improved memory, learning, and cognitive function. This makes it essential for research on neuronal development and plasticity.

• Protease Inhibition: Neuroserpin preferentially inhibits tPA, modulating extracellular proteolysis in the brain. This is critical for investigating the balance between protease activity and inhibition in processes such as neuronal migration, axonal growth, and synaptic remodeling.

• Disease Modeling: Changes in neuroserpin expression and activity are linked to pathologies such as Alzheimer’s disease, familial encephalopathy with neuroserpin inclusion bodies (FENIB), cancer metastasis, and glaucoma. Recombinant neuroserpin enables mechanistic studies and drug screening for these conditions.

• Cellular Signaling and Apoptosis: Neuroserpin modulates apoptotic pathways, including the expression of Bcl-2 and caspase-3, and influences signaling cascades such as MAPK and NFκB, relevant for research on cell survival and stress responses.

• Controlled Experimental Conditions: Recombinant protein allows for precise dosing, purity, and reproducibility in in vitro and in vivo experiments, facilitating mechanistic studies and reducing variability compared to endogenous sources.

In summary, recombinant human neuroserpin is a versatile tool for investigating neurobiology, neuroprotection, synaptic function, and disease mechanisms, as well as for screening potential therapeutic interventions targeting serpin-related pathways.

Recombinant Human Neuroserpin can be used as a standard for quantification or calibration in ELISA assays, provided it is of high purity, its concentration is accurately determined, and it is compatible with your assay’s antibodies and detection system. This is a common practice in quantitative ELISA protocols for neuroserpin and other proteins.

Key considerations and supporting details:

• Standard Curve Preparation: Most commercial human neuroserpin ELISA kits use recombinant human neuroserpin as the standard to generate a calibration curve, against which unknown sample concentrations are measured. The standard is serially diluted to cover the assay’s dynamic range, and the resulting curve is used for quantification.

• Purity and Quantification: The recombinant neuroserpin used as a standard should be of high purity (typically >80% or higher) and its concentration must be accurately determined, usually by absorbance at 280 nm or amino acid analysis. Impurities or inaccurate quantification can introduce error into your assay.

• Epitope Recognition: The antibodies used in your ELISA must recognize epitopes present on the recombinant neuroserpin standard. Most ELISA kits are validated to detect both recombinant and native neuroserpin, but if you are developing your own assay, confirm antibody compatibility.

• Matrix Effects: When preparing your standard curve, dilute the recombinant neuroserpin in the same buffer or matrix as your samples (e.g., serum, plasma, or assay buffer) to minimize matrix effects and ensure accurate quantification.

• Assay Validation: If you are using a recombinant standard not provided with a commercial kit, validate the assay’s linearity, sensitivity, and recovery using your standard to ensure reliable quantification.

• Documentation: Refer to the instructions for use (IFU) or protocol booklet of your ELISA kit for specific recommendations regarding standard preparation and compatibility.

Summary Table: Use of Recombinant Human Neuroserpin as ELISA Standard

In conclusion, recombinant human neuroserpin is suitable as a standard for ELISA quantification if these best practices are followed. Always validate performance in your specific assay context.

Recombinant Human Neuroserpin has been validated in published research for several key applications, primarily in neuroscience and cell biology:

• Neuroprotection assays: Recombinant neuroserpin has been used to protect primary hippocampal neurons from oxidative stress (e.g., H₂O₂-induced damage), oxygen-glucose deprivation, and excitotoxicity. These studies assess cell viability, apoptosis (e.g., Bcl-2 and caspase-3 expression), and LDH release as endpoints.
• Synaptic plasticity and neuronal development: It has been applied to hippocampal neuron cultures to study effects on dendritic spine density, neurite outgrowth, and synaptic maturation, indicating roles in axogenesis and synaptic modeling.
• Ischemia and stroke models: Exogenous administration in animal models has been shown to reduce cerebral infarct volume and increase neuronal survival after ischemic or embolic injury.
• Cell signaling studies: Recombinant neuroserpin has been used to dissect its influence on signaling pathways, such as MAPK, NFκB, and TNFα, in the context of neuroinflammation and neurodegeneration.
• Protein trafficking and polymerization studies: Both wild-type and mutant forms (e.g., G392E) of recombinant neuroserpin have been used to investigate secretion pathways, ER-to-Golgi trafficking, and polymerization behavior in cell models (e.g., HEK-293 cells, primary neurons).
• Disease modeling: Recombinant neuroserpin has been used to model and study mechanisms underlying diseases such as Familial Encephalopathy with Neuroserpin Inclusion Bodies (FENIB), Alzheimer’s disease, and glaucoma, including its effects on neurovascular permeability and cancer metastasis.

Common experimental techniques and endpoints include:

• Cell viability assays (e.g., MTT, LDH release)
• Western blotting for protein expression and polymerization status
• Immunocytochemistry for synaptic markers (e.g., PSD-95)
• Animal models for neuroprotection and infarct size assessment
• Analysis of apoptotic and anti-apoptotic protein expression

These applications demonstrate that recombinant human neuroserpin is a versatile tool for studying neuroprotection, synaptic biology, protein trafficking, and disease mechanisms in both in vitro and in vivo systems.

To reconstitute and prepare Recombinant Human Neuroserpin protein for cell culture experiments, follow these steps:

• Centrifuge the vial briefly to ensure all lyophilized protein is at the bottom before opening.
• Reconstitute the protein in sterile distilled water (pH 8.0) to achieve a concentration of approximately 0.1 mg/mL. Alternatively, you may use an aqueous buffer containing 0.1% BSA (bovine serum albumin) if additional protein stabilization is desired.
• Mix gently by pipetting up and down or by gentle vortexing to ensure complete dissolution.
• If a higher concentration is needed, adjust the volume of water or buffer accordingly, typically within the range of 0.1–1.0 mg/mL.
• Aliquot the reconstituted protein into working volumes to avoid repeated freeze-thaw cycles, which can denature the protein.
• Storage: Store aliquots at -20°C to -80°C for long-term use. For short-term use (up to one week), storage at 4°C is acceptable.
• Before adding to cell culture, ensure the protein solution is sterile. If necessary, filter-sterilize using a 0.22 μm filter.

Additional notes for cell culture:

• If using directly in cell culture, consider diluting the reconstituted protein in your cell culture medium or a compatible buffer immediately before use.
• The presence of carrier proteins (e.g., BSA) can help prevent adsorption to plasticware and improve stability, especially at low working concentrations.
• Always consult the specific product datasheet or certificate of analysis for any additional or product-specific recommendations.

Summary of key steps:

These steps will help ensure that recombinant human Neuroserpin is properly prepared for reliable and reproducible cell culture experiments.