Recombinant Mouse GDNF

Recombinant Mouse GDNF (Glial cell line-derived neurotrophic factor) is widely used in research because it is a potent neurotrophic factor that promotes the survival, differentiation, and maturation of various neuronal populations, especially dopaminergic and motor neurons, and confers neuroprotection in models of neurodegeneration and injury.

Key scientific applications and rationale include:

• Neuronal Survival and Differentiation: Recombinant mouse GDNF enhances the survival and morphological differentiation of dopaminergic neurons, increases high-affinity dopamine uptake, and promotes the proliferation and maturation of motor neuron precursors. This makes it essential for studies on neuronal development, neurodegenerative diseases, and regenerative medicine.

• Neuroprotection: GDNF protects neurons from toxic insults and excitotoxicity, such as those induced by kainate, and has been shown to rescue motor neurons from programmed cell death and axonal injury in various animal models. This property is particularly valuable in research on Parkinson’s disease, amyotrophic lateral sclerosis (ALS), and spinal cord injury.

• Functional Enhancement: Elevated GDNF levels in mice lead to increased dopamine levels, improved dopamine transporter activity, and enhanced motor coordination without adverse behavioral effects. Recombinant GDNF can be used to model these effects in vitro and in vivo.

• Synaptic and Electrophysiological Maturation: GDNF promotes axonal sprouting, enhances neurotransmitter release, and increases electrophysiological maturity of neurons, which is critical for studies on synaptic development and function.

• Disease Modeling and Therapeutic Research: Recombinant mouse GDNF is used to investigate mechanisms and potential treatments for neurodegenerative disorders, addiction, ischemic stroke, and other conditions associated with neuronal death.

• Experimental Control and Reproducibility: Using recombinant mouse GDNF ensures species-specific activity and consistency in experimental protocols, which is crucial for reproducible results in mouse-based cell culture and animal studies.

Best Practices:

• Recombinant mouse GDNF should be used at empirically determined concentrations, typically in the range of 10–100 ng/mL for cell culture applications, depending on the cell type and experimental goals.
• It is important to include appropriate controls, such as neutralizing antibodies or vehicle treatments, to validate the specificity of GDNF’s effects.

In summary, recombinant mouse GDNF is a critical tool for neuroscience research, enabling precise investigation of neuronal survival, differentiation, neuroprotection, and functional maturation in mouse models and cell cultures.

Yes, recombinant mouse GDNF can be used as a standard for quantification or calibration in ELISA assays, provided it is properly validated for your specific assay system. Recombinant mouse GDNF is commonly used as a standard in commercial mouse GDNF ELISA kits, where it enables the generation of a standard curve for quantifying GDNF concentrations in biological samples.

Key considerations and supporting details:

• Standard Curve Generation: ELISA kits for mouse GDNF typically include a recombinant mouse GDNF standard, which is serially diluted to create a standard curve. This curve is used to interpolate the concentration of GDNF in unknown samples.
• Assay Compatibility: The recombinant standard should match the species and isoform of GDNF you intend to measure. Most mouse GDNF ELISA kits are designed to detect the murine form and use recombinant mouse GDNF produced in E. coli as the standard.
• Validation: If you are using a recombinant mouse GDNF standard outside of a commercial kit, ensure that:
  • The recombinant protein is of high purity and correctly folded.
  • The concentration is accurately determined (e.g., by BCA or Bradford assay).
  • The standard is prepared and diluted in the same buffer as your samples to minimize matrix effects.
  • The antibodies in your ELISA recognize the recombinant form equivalently to the native protein.
• Research Use Only: Most recombinant standards and ELISA kits are for research use only and not for diagnostic or clinical applications.
• Expression System: Recombinant mouse GDNF is often produced in E. coli, which may lack post-translational modifications present in native GDNF. This is generally acceptable for ELISA calibration, but if your assay is sensitive to such modifications, consider this factor.

Protocol best practices:

• Reconstitute the recombinant standard according to the manufacturer’s instructions, using the recommended diluent and procedure to ensure consistency and accuracy.
• Prepare a serial dilution series to cover the expected range of GDNF concentrations in your samples.
• Always run the standard curve in parallel with your samples for each assay plate.

Limitations:

• If your ELISA is designed for a different species or uses antibodies with limited cross-reactivity, confirm that the recombinant mouse GDNF is suitable as a standard for your assay.
• Some kits or protocols may recommend against using recombinant protein as a standard for certain applications; always consult the specific kit documentation.

In summary, recombinant mouse GDNF is widely accepted and used as a standard for ELISA quantification, provided it is validated for your assay and used according to best practices.

Recombinant Mouse GDNF has been validated for several key applications in published research, primarily focusing on its neurotrophic activity and utility in neurobiology and disease models.

Validated Applications:

• Bioactivity Assays: Recombinant Mouse GDNF is routinely used to assess its ability to promote survival, differentiation, and dopamine uptake in dopaminergic neurons, often measured by dose-dependent proliferation of cell lines such as C6 cells.
• Functional Studies (FuncS): It is applied in functional assays to study neuronal survival, morphological differentiation, and neuroprotection, particularly in models of neurodegenerative diseases like Parkinson’s disease.
• Western Blot Control: GDNF is used as a positive control in Western blotting to validate antibody specificity and protein detection.
• SDS-PAGE: The protein is validated for purity and integrity using SDS-PAGE analysis.
• Neutralization Assays: It serves as a target in neutralization experiments to test the efficacy of antibodies or inhibitors against GDNF.
• In Vivo Disease Models: Recombinant Mouse GDNF has been administered in mouse models to study its therapeutic effects, especially in Parkinson’s disease, where it demonstrates neuroprotective and motor function benefits.

Supporting Details:

• Neurotrophic Factor: GDNF enhances survival and morphological differentiation of dopaminergic neurons and increases high-affinity dopamine uptake by binding to its coreceptor GFRA1 and activating the RET receptor.
• Developmental Biology: It is involved in neural crest development and has been used to study kidney and testis development in mice.
• Therapeutic Research: Applications include investigating its potential in treating neurodegenerative diseases such as Parkinson’s and Huntington’s disease.

Summary Table of Validated Applications

These applications are supported by both product validation data and peer-reviewed research, confirming the utility of recombinant Mouse GDNF in neurobiology, disease modeling, and protein analysis.

To reconstitute and prepare Recombinant Mouse GDNF protein for cell culture experiments, dissolve the lyophilized protein in sterile water to a concentration of at least 100 μg/mL, then dilute further in appropriate buffer or culture medium as needed for your assay.

Detailed protocol and best practices:

• Reconstitution:

  • Add sterile, ultrapure water (18 MΩ-cm) directly to the lyophilized GDNF powder to achieve a concentration of at least 100 μg/mL.
  • Gently swirl or invert the vial to mix. Do not vortex, as this may denature the protein.
  • Allow several minutes for complete dissolution.
  • If a different concentration is required, adjust the volume accordingly, but avoid concentrations below 100 μg/mL to maintain protein stability.

• Dilution for cell culture:

  • After initial reconstitution, dilute the stock solution to your desired working concentration using sterile cell culture medium or buffer.
  • For most cell-based assays, GDNF is typically used in the range of 1–100 ng/mL, but optimal concentrations should be determined empirically for your specific application.

• Carrier protein (optional but recommended for stability):

  • To minimize adsorption and loss of activity, especially at low concentrations, dilute GDNF in buffer containing 0.1% BSA or another suitable carrier protein.
  • This is particularly important if you plan to store aliquots for future use.

• Storage:

  • After reconstitution, the stock solution can be stored at 4°C for up to 2–7 days.
  • For longer-term storage, aliquot and freeze at –20°C to –80°C.
  • Avoid repeated freeze-thaw cycles, as these can reduce protein activity.
  • For maximum stability, store aliquots in the presence of a carrier protein.

• General notes:

  • Always use sterile technique to avoid contamination.
  • Confirm the absence of additives in the lyophilized product; if none are present, reconstitution in water is appropriate.
  • If your application is sensitive to endotoxin, verify the endotoxin level of your preparation (typically <1 EU/μg for high-quality recombinant GDNF).

Summary Table: Recombinant Mouse GDNF Reconstitution

These guidelines are based on standard protocols for recombinant GDNF and should be adapted as needed for your specific experimental requirements.