Recombinant Human GFRα-1

Recombinant Human GFRα-1 is used in research applications to study and manipulate the GDNF (glial cell line-derived neurotrophic factor) signaling pathway, which is critical for neuronal survival, development, and regeneration, as well as for investigating cancer cell migration and stem cell biology.

Key scientific reasons to use recombinant human GFRα-1 include:

• Essential Co-receptor for GDNF Signaling: GFRα-1 acts as a high-affinity co-receptor for GDNF, forming a complex with the RET tyrosine kinase to initiate downstream signaling. This pathway is vital for the survival and maintenance of dopaminergic neurons, kidney development, and other neurobiological processes.

• Functional Studies in Cell Culture and Bioassays: Recombinant GFRα-1 enables controlled activation of GDNF/RET signaling in vitro, allowing researchers to dissect molecular mechanisms, assess cell survival, differentiation, and migration, and model disease states such as Parkinson’s disease and Hirschsprung’s disease.

• Soluble Form Enhances Experimental Flexibility: The recombinant, soluble form of GFRα-1 can be added exogenously to cell cultures, enabling studies where endogenous GFRα-1 is absent or needs to be precisely titrated. This is particularly useful for investigating dose-dependent effects on cell migration, MAPK pathway activation, and cancer cell perineural invasion.

• Stem Cell and Regenerative Medicine Applications: GFRα-1 is involved in the self-renewal and maintenance of spermatogonial stem cells and hematopoietic stem cells, making recombinant protein valuable for protocols aiming to expand or differentiate stem cell populations.

• Cancer Biology Research: Soluble GFRα-1 has been shown to cooperate with GDNF to enhance cancer cell migration and perineural invasion via RET activation, providing a tool to study tumor microenvironment interactions and metastatic mechanisms.

• Specificity and Selectivity: Recombinant human GFRα-1 is highly selective for GDNF over other ligands such as artemin, ensuring targeted activation of the desired signaling pathway in experimental systems.

Typical applications include:

• Bioassays to measure GDNF activity and RET phosphorylation.
• Cell culture experiments to study neuronal survival, differentiation, and migration.
• Stem cell maintenance and expansion protocols.
• Cancer cell migration and invasion assays.
• ELISA standards for quantifying GFRα-1 or GDNF in biological samples.

In summary, recombinant human GFRα-1 is a critical reagent for dissecting GDNF/RET signaling, modeling neurodevelopmental and neurodegenerative diseases, studying stem cell biology, and investigating cancer cell behavior in a controlled and reproducible manner.

Yes, you can use Recombinant Human GFRα-1 as a standard for quantification or calibration in ELISA assays, provided that the recombinant protein is suitable for your specific assay format and detection system.

Key Considerations:

• Purity and Formulation: Ensure the recombinant GFRα-1 is highly purified and formulated appropriately (e.g., with or without carrier proteins like BSA, depending on your assay requirements). For ELISA standards, it is generally recommended to use the recombinant protein without carrier proteins to avoid interference.

• Compatibility: The recombinant protein should be compatible with the antibodies used in your ELISA. It should be recognized by both the capture and detection antibodies to ensure accurate quantification.

• Validation: Validate the recombinant protein as a standard by running a dilution series to confirm linearity and reproducibility within the expected concentration range of your samples.

• Application Note: Some suppliers specifically recommend their recombinant proteins for use as ELISA standards. For example, R&D Systems advises using recombinant proteins with BSA for cell or tissue culture, but for ELISA standards, the protein without BSA is preferred.

References:

• R&D Systems: Recommends using recombinant protein without BSA for ELISA standards.
• BioLegend: Notes that ELISA standard recombinant proteins are not recommended for bioassay usage but are suitable for ELISA calibration.

By following these guidelines, you can effectively use Recombinant Human GFRα-1 as a standard in your ELISA assays for accurate quantification and calibration.

Recombinant Human GFRα-1 (GFRA1) has been validated for several key applications in published research, primarily related to its role as a coreceptor in GDNF signaling and its utility in functional assays. The main applications supported by the literature include:

1. Bioassays and Functional Assays

   • Used to study GDNF-mediated signaling, including activation of RET and downstream pathways such as ERK1/2 MAPK and Akt phosphorylation.
   • Employed in luciferase reporter assays to measure GDNF biological activity in neural cells cotransfected with RET and reporter constructs.
   • Validated for measuring cell migration in response to GDNF, particularly in cancer cell lines and neuronal models.

2. ELISA and Binding Assays

   • Applied as a capture or detection reagent in ELISA to assess binding affinity of GDNF or GDNF fusion proteins (e.g., cTfRMAb-GDNF) to GFRα1.
   • Used to confirm the specificity and potency of GDNF family ligands and their fusion proteins.

3. Cell Culture Studies

   • Utilized in cell culture systems to investigate the effects of GDNF/GFRα1 signaling on cell survival, differentiation, and migration.
   • Employed in studies of dopaminergic neuron survival and neuroprotection.

4. Neuroscience and Disease Models

   • Applied in studies of Parkinson’s disease and neurodegeneration, where GDNF/GFRα1 signaling is critical for dopaminergic neuron maintenance.
   • Used in models of perineural invasion in cancer to study the role of soluble GFRα1 in enhancing GDNF-mediated migration and signaling.

5. Protein-Protein Interaction Studies

   • Used in studies of synaptic adhesion and receptor clustering, including validation of GFRα1:GFRα1 pentameric interfaces.

In summary, Recombinant Human GFRα-1 has been validated for use in bioassays, ELISA, cell culture, neuroprotection studies, cancer migration assays, and protein interaction studies, making it a versatile tool for investigating GDNF signaling and its biological effects.

Reconstitution Protocol

Recombinant Human GFRα-1 proteins are typically supplied in lyophilized form and require proper reconstitution before use in cell culture experiments. Begin by centrifuging the vial for 20-30 seconds in a microcentrifuge before opening. This step ensures that any protein adhered to the cap or tube walls is driven to the bottom of the vial, maximizing recovery of your product.

Reconstitution Procedure

Diluent Selection and Volume

Reconstitute the lyophilized protein using sterile, distilled water or PBS, depending on your specific product formulation. The standard reconstitution concentration range is 0.1 to 1.0 mg/mL. For example, if you have 100 µg of protein, add between 100 µL and 1 mL of diluent to achieve the desired concentration. Typical reconstitution volumes are approximately 100 µL for 10-50 µg vials and 1 mL for 100 µg vials.

Reconstitution Technique

Add the recommended volume of sterile diluent to the vial and gently shake to solubilize the protein completely. Do not vortex, as vigorous mixing can cause foaming and protein denaturation. Allow the vial to reconstitute for 15-30 minutes at room temperature with gentle agitation.

Optional Additives

For enhanced stability and extended storage capability, consider adding 5-50% glycerol (final concentration) to your reconstituted solution. This cryoprotectant helps preserve protein integrity during freeze-thaw cycles.

Storage and Stability

After reconstitution, store your GFRα-1 protein under the following conditions:

• Short-term storage: 1 month at 2-8 °C under sterile conditions
• Long-term storage: 3 months at -20 to -70 °C under sterile conditions

Critical handling recommendations include using a manual defrost freezer and avoiding repeated freeze-thaw cycles. Aliquot the reconstituted protein into smaller portions to minimize exposure to freeze-thaw stress during future use.

Verification and Quality Control

Before proceeding with cell culture experiments, verify protein integrity by checking the certificate of analysis provided with your shipment, which contains specific reconstitution instructions tailored to your particular product formulation.