Recombinant Human GFRα-3

Recombinant Human GFRα-3 is primarily used in research applications to study neurotrophic signaling, neuronal survival, and pain mechanisms, particularly those involving the GDNF family receptor alpha 3 (GFRA3) and its ligand artemin.

Key scientific reasons to use recombinant human GFRα-3 include:

• Modeling Neurotrophic Signaling: GFRα-3 is a glycosylphosphatidylinositol (GPI)-linked cell surface receptor that forms a signaling complex with the RET tyrosine kinase receptor. It preferentially binds artemin, a neurotrophic factor, enabling detailed studies of the GFRα3-RET signaling pathway in vitro.

• Neuronal Survival and Differentiation: The GFRα3-ARTN-RET axis is critical for the survival, migration, and differentiation of specific neuronal populations, such as those in the dorsal root ganglion and superior cervical ganglion. Recombinant GFRα-3 allows for controlled experiments on neuronal cultures to assess neuroprotective effects and neurite outgrowth.

• Pain Mechanisms and Nociception: GFRα-3 is highly expressed in nociceptive neurons. Studies using recombinant GFRα-3 have elucidated its role in pain signaling and chronic pain models, making it valuable for research into analgesic drug development and pain pathway modulation.

• Protein-Protein Interaction Studies: Recombinant GFRα-3 is used in binding assays (e.g., surface plasmon resonance) to characterize interactions with artemin, RET, and other potential partners, facilitating mechanistic studies and screening of therapeutic candidates.

• Antibody Development and Screening: Recombinant GFRα-3 serves as an antigen for generating and screening monoclonal antibodies targeting GFRα-3, which are being investigated for their potential to modulate pain and other neurobiological processes.

• Cellular and Molecular Assays: It is suitable for use as a standard in ELISA, Western blot, and other immunoassays to quantify GFRα-3 expression or activity in biological samples.

Best practices for using recombinant human GFRα-3:

• Select the appropriate format (e.g., Fc-tagged, carrier-free) based on your assay requirements.
• Store at recommended conditions (typically -70°C) to maintain protein stability.
• Use in combination with artemin or RET for functional assays to recapitulate physiological signaling.

In summary, recombinant human GFRα-3 is a versatile tool for dissecting neurotrophic factor signaling, investigating pain pathways, and developing targeted therapeutics in neuroscience and cell biology research.

Yes, you can use recombinant Human GFRα-3 as a standard for quantification or calibration in your ELISA assays, provided it is sufficiently pure and its concentration is accurately determined. Recombinant proteins are commonly used as standards in quantitative ELISA protocols to generate standard curves for analyte quantification.

Key considerations and best practices:

• Purity and Identity: The recombinant Human GFRα-3 should be highly purified and correctly folded to ensure it mimics the native protein's immunoreactivity.
• Concentration Determination: Accurately measure the protein concentration, ideally using a method such as HPLC or UV absorbance, as errors in concentration will directly affect quantification accuracy.
• Standard Curve Preparation: Prepare serial dilutions of the recombinant protein to cover the expected range of analyte concentrations in your samples. Typical standard curve ranges for GFRα-3 ELISA kits are from low picogram to nanogram per milliliter concentrations (e.g., 16–4000 pg/mL).
• Validation: Confirm that the recombinant standard is recognized by the antibodies used in your ELISA. Most commercial kits specify that they detect both natural and recombinant forms of the protein.
• Lot-to-Lot Variation: Be aware that different lots of recombinant protein may show slight differences in immunoreactivity, so it is best to assign values based on ELISA measurement rather than relying solely on the mass stated on the vial.

Protocol notes:

• Always run a standard curve with each assay to ensure accurate quantification.
• Follow manufacturer or published protocols for reconstitution and dilution of the recombinant standard, as these may be lot-specific.
• Validate your standard curve by comparing calculated concentrations with known spiked samples, if possible.

Limitations:

• If the recombinant protein is not full-length or lacks post-translational modifications present in native samples, there may be differences in antibody recognition.
• For regulatory or diagnostic applications, additional validation may be required.

In summary, recombinant Human GFRα-3 is suitable as a standard for ELISA quantification if you ensure purity, accurate concentration, and immunoreactivity with your assay system.

Recombinant Human GFRα-3 has been validated for several key applications in published research, primarily in studies of neurobiology, pain mechanisms, and disease pathology.

Validated Applications:

• Western Blot (WB): Recombinant Human GFRα-3 has been used as a positive control and for detection of GFRα-3 expression in tissue samples, including studies comparing normal and diseased states such as chronic pancreatitis.
• Enzyme-Linked Immunosorbent Assay (ELISA): The protein is suitable for use as a standard or antigen in ELISA assays to quantify GFRα-3 levels in biological samples.
• Immunocytochemistry/Immunofluorescence: GFRα-3 has been used for double immunofluorescence labeling to study its co-localization with other markers in tissue sections, particularly in neural and pancreatic tissues.
• Antibody Screening and Validation: Recombinant GFRα-3 has been used to generate and screen monoclonal antibodies, including high-affinity blocking antibodies for preclinical and clinical studies targeting pain pathways.
• Cell-based Functional Assays: It has been employed in luciferase reporter assays to assess artemin-mediated GFRα-3 signaling in transfected cell lines (e.g., HEK293 cells expressing GFRα-3 and RET).
• Preclinical Animal Models: Recombinant GFRα-3 has been used to study pain mechanisms, such as tactile allodynia and hyperalgesia, by evaluating the efficacy of anti-GFRα-3 antibodies in mouse models of osteoarthritis-like and inflammatory pain.
• Disease Pathology Research: Increased expression of GFRα-3 has been validated in disease models, such as chronic pancreatitis, using Western blot and immunohistochemistry.

Additional Context:

• Neurobiology: GFRα-3 is highly expressed in dorsal root ganglion neurons, particularly nociceptors, and is involved in artemin signaling, which influences neuron survival and pain sensitivity.
• Drug Target Validation: Recombinant GFRα-3 is used in drug discovery and validation studies, especially for developing therapeutics targeting the artemin-GFRα-3 pathway in pain and neurodegenerative conditions.

Summary Table of Validated Applications

These applications are supported by peer-reviewed studies and are commonly used in both basic and translational research involving GFRα-3.

To reconstitute and prepare Recombinant Human GFRα-3 protein for cell culture experiments, dissolve the lyophilized protein in sterile, distilled or deionized water to achieve a concentration between 0.1–1.0 mg/mL. The exact concentration should be chosen based on your experimental requirements and the solubility of the protein.

Step-by-step protocol:

1. Centrifuge the vial briefly before opening to ensure all lyophilized material is at the bottom.
2. Add sterile water (or PBS if specified by the product datasheet) to the vial. For most applications, a concentration of 0.25 mg/mL is commonly used, but concentrations between 0.1–1.0 mg/mL are typical.
3. Gently mix by swirling or inverting. Avoid vigorous shaking or vortexing to prevent protein denaturation or foaming.
4. Allow the protein to dissolve at room temperature for 15–30 minutes with gentle agitation.
5. Aliquot the reconstituted solution into smaller volumes to avoid repeated freeze-thaw cycles, which can degrade the protein.
6. Storage after reconstitution:
   • Short-term: Store at 2–8°C for up to one week.
   • Long-term: Store aliquots at –20°C to –80°C for up to several months.

Additional recommendations:

• If the protein is to be used in cell culture, ensure the final buffer is compatible with your cells (e.g., PBS, pH 7.2–7.5, without calcium or magnesium, is common).
• For sensitive applications, consider adding a carrier protein (e.g., 0.1% BSA) or glycerol (5–50%) to stabilize the protein during storage.
• Always check the product-specific datasheet for any unique requirements regarding reconstitution buffer, concentration, or additives.

Critical notes:

• Avoid repeated freeze-thaw cycles to maintain protein integrity.
• Use aseptic technique throughout to prevent contamination, especially for cell culture applications.

This protocol is based on standard practices and multiple product datasheets for recombinant human GFRα-3 protein. Always refer to the specific datasheet provided with your protein for any lot-specific instructions.