Recombinant Human GFRα-2

Recombinant Human GFRα-2 is used in research applications to study neurotrophic signaling, neuronal differentiation, and neuroplasticity, particularly as a co-receptor for the RET tyrosine kinase in response to ligands such as neurturin (NRTN) and glial cell line-derived neurotrophic factor (GDNF).

Key scientific applications and advantages:

• Neurotrophic Signaling Studies: GFRα-2 is a critical component of the NRTN/GDNF signaling axis, which regulates neuronal survival, differentiation, and plasticity. Recombinant GFRα-2 allows researchers to dissect the molecular mechanisms underlying these processes, including activation of downstream pathways such as ERK1/2 and Akt.

• Neuronal Differentiation and Outgrowth: Expression of GFRα-2 isoforms in neuronal cell models (e.g., Neuro2A cells) enables investigation of their distinct roles in neurite outgrowth and inhibition, as well as their interaction with other neurotrophic receptors. For example, GFRα-2b has been shown to exert a dominant inhibitory effect on neurite outgrowth, highlighting the importance of isoform-specific studies.

• Neuroplasticity and Disease Modeling: The NRTN/GFRα-2 axis is implicated in neuroplastic changes during chronic diseases such as chronic pancreatitis, where increased GFRα-2 expression correlates with reactive neural alterations. Recombinant GFRα-2 can be used to model these changes in vitro and to test therapeutic interventions targeting neurotrophic pathways.

• Developmental Biology: GFRα-2 is expressed in various regions of the human brain and is involved in the development of neural structures. Recombinant protein enables functional assays to study its role in CNS development and regional expression patterns.

• Protein Interaction and Binding Studies: Recombinant GFRα-2 is suitable for biochemical assays such as ELISA, Western blot, and receptor-ligand binding studies, facilitating the characterization of its interactions with GDNF family ligands and RET.

Best practices for use:

• Select the appropriate isoform or fragment for your experimental model, as different isoforms may have distinct biological activities.
• Use recombinant GFRα-2 in combination with its ligands (e.g., NRTN, GDNF) and RET to reconstitute signaling complexes in cell-based or biochemical assays.
• Validate expression and activity using sensitive detection methods such as real-time PCR, Western blot, or phosphorylation assays.

Summary:
Using recombinant human GFRα-2 enables precise mechanistic studies of neurotrophic signaling, neuronal differentiation, and neuroplasticity, and supports disease modeling and developmental biology research. Its recombinant form ensures reproducibility and specificity in functional assays and protein interaction studies.

Recombinant Human GFRα-2 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 approach is widely accepted for generating standard curves in quantitative ELISA protocols.

Essential context and best practices:

• Purity and Quantification: The recombinant protein should be highly purified, ideally verified by SDS-PAGE and HPLC, and its concentration must be accurately measured (e.g., by absorbance at 280 nm or BCA assay).
• Standard Curve Preparation: Prepare serial dilutions of the recombinant GFRα-2 to cover the expected concentration range in your samples. The standard curve should be run in parallel with your samples in each assay to ensure accurate quantification.
• Validation: Confirm that the recombinant GFRα-2 behaves similarly to endogenous GFRα-2 in your assay matrix (e.g., serum, plasma, cell culture supernatant). Parallelism between the standard curve and sample dilution curves is essential for reliable quantification.
• Format Compatibility: Ensure the recombinant GFRα-2 is compatible with your ELISA format (e.g., sandwich or direct ELISA) and that the antibodies used in the assay recognize both recombinant and native forms.
• Research Use: Most recombinant standards are intended for research use only and not for diagnostic applications.

Additional relevant information:

• Carrier Proteins: Some recombinant proteins are supplied with carrier proteins (e.g., BSA) to enhance stability. If using such formulations, account for the carrier in your calculations and assay design.
• Lot-to-Lot Consistency: Always use the same lot of recombinant protein for calibration within a study to minimize variability.
• Documentation: Record the source, lot number, and preparation details of your recombinant standard for reproducibility and troubleshooting.

In summary, using recombinant Human GFRα-2 as an ELISA standard is scientifically valid and commonly practiced, provided you follow rigorous preparation, validation, and documentation protocols.

Recombinant Human GFRα-2 has been validated in published research primarily for studies involving neuronal differentiation, neuroplasticity, and neurotrophic signaling, particularly in the context of the GDNF family ligand-receptor system.

Key validated applications include:

• Neuronal Differentiation and Signaling Assays
  Recombinant GFRα-2 has been used to study its role in neuronal differentiation models, such as in Neuro2A and BE(2)-C cell lines. These studies examined how different GFRα-2 isoforms affect neurite outgrowth, ERK1/2 and Akt phosphorylation, and RhoA activation in response to ligands like GDNF and neurturin (NTN). This application is central to understanding the molecular mechanisms of neurotrophic factor signaling.

• Neuroplasticity Assays
  In vitro neuroplasticity assays have utilized recombinant human GFRα-2 to investigate its involvement in neural alterations, such as those occurring in chronic pancreatitis. For example, dorsal root ganglion (DRG) neurons cultured with recombinant neurturin (NRTN) and GFRα-2 were used to assess changes in neuronal and glial cell populations, demonstrating the neurotrophic and neuroplastic effects mediated by the NRTN/GFRα-2 axis.

• Expression and Localization Studies
  Recombinant GFRα-2 has been used as a standard or control in immunohistochemistry and Western blot assays to detect endogenous GFRα-2 in tissues, including human brain and pancreas. These studies help map the distribution and expression levels of GFRα-2 in various physiological and pathological contexts.

• RET Co-receptor Functional Studies
  Research has validated recombinant GFRα-2 in studies of RET receptor signaling, examining its co-expression and functional interaction with other GFRα family members in tissues such as the pituitary gland. This application is important for understanding the broader role of GFRα-2 in neuroendocrine regulation.

Additional relevant contexts:

• Cancer Biology: Systematic reviews highlight the emerging role of GFRα family members, including GFRα-2, in cancer biology, suggesting recombinant GFRα-2 may be used in functional assays to study tumor cell signaling and behavior.
• Developmental Biology: Recombinant GFRα-2 is implicated in studies of nervous system development, particularly in the formation of parasympathetic innervation and myenteric nerve plexus.

Summary Table: Validated Applications of Recombinant Human GFRα-2

These applications are supported by published research and demonstrate the utility of recombinant human GFRα-2 in both basic and translational neuroscience, neuroendocrinology, and disease modeling.

To reconstitute and prepare Recombinant Human GFRα-2 protein for cell culture experiments, follow these best-practice steps:

1. Centrifuge the vial briefly at 4°C before opening to ensure all lyophilized powder is at the bottom and not lost upon opening.

2. Reconstitution buffer:

   • Use sterile, deionized water or 10 mM PBS (pH 7.4) as the solvent, unless your product datasheet specifies otherwise.
   • For most cell culture applications, PBS is preferred for physiological compatibility.

3. Protein concentration:

   • Reconstitute to a final concentration of 0.1–1.0 mg/mL.
   • Example: For 100 µg of protein, add 100 µL (for 1 mg/mL) to 1 mL (for 0.1 mg/mL) of buffer.

4. Dissolution:

   • Gently swirl or invert the vial to dissolve the protein. Avoid vigorous shaking or vortexing to prevent denaturation or foaming.
   • If the protein does not dissolve immediately, allow it to sit on ice or at 4°C for up to 2 hours, gently mixing occasionally.

5. Carrier protein (optional but recommended):

   • To minimize adsorption and stabilize the protein, especially at low concentrations, add a carrier protein such as 0.1% BSA, 10% FBS, or 5% HSA to your buffer.
   • For serum-free or in vivo applications, use trehalose instead of animal-derived carriers.

6. Aliquoting and storage:

   • After reconstitution, aliquot the solution to avoid repeated freeze-thaw cycles.
   • Store aliquots at 2–8°C for up to 1 week for short-term use, or at –20°C to –80°C for long-term storage (up to several months).
   • For long-term storage, adding 5–50% glycerol can further stabilize the protein.

7. Preparation for cell culture:

   • Thaw aliquots on ice and dilute to the desired working concentration in your cell culture medium immediately before use.
   • If using serum-free medium, ensure no animal-derived carrier proteins are present in your final dilution.

Summary Table: Reconstitution and Storage

Note: Always consult the specific Certificate of Analysis (CoA) or product datasheet for your recombinant GFRα-2, as some preparations may have unique requirements.

Key technical tips:

• Avoid repeated freeze-thaw cycles to preserve protein activity.
• Use low-protein binding tubes for dilution and storage.
• For functional assays, confirm protein activity post-reconstitution if possible.

If you need a protocol tailored to a specific application (e.g., neuronal differentiation, RET signaling assays), please specify.