Recombinant Rat CNTF Rα

Using Recombinant Rat CNTF Rα (Ciliary Neurotrophic Factor Receptor Alpha) in research is essential for studying and enhancing the biological activity of CNTF, particularly in systems where endogenous CNTF receptor expression is limited or where precise control of CNTF signaling is required.

Key reasons to use Recombinant Rat CNTF Rα in research applications:

• Facilitates CNTF Signaling in Non-Responsive Cells: Many cell types do not naturally express sufficient levels of CNTF receptor alpha (CNTFRα), which is required for CNTF to bind and activate downstream signaling pathways. Adding recombinant CNTFRα can render these cells responsive to CNTF, enabling studies of CNTF’s effects in a broader range of cell types.

• Enhances Biological Activity of CNTF: Soluble CNTFRα (sCNTFRα) can form a complex with CNTF, which then interacts with the gp130/LIFRβ signaling complex on target cells, amplifying CNTF’s neuroprotective and trophic effects. This is particularly important in retinal and neuronal studies where indirect effects via glial cells (e.g., Müller cells) are significant.

• Modeling Disease and Neuroprotection: In retinal degeneration and neurodegenerative disease models, exogenous CNTF (often delivered as recombinant protein) is used to promote photoreceptor and neuron survival. Recombinant CNTFRα allows for controlled, reproducible activation of CNTF pathways, which is critical for dissecting mechanisms of neuroprotection and for preclinical therapeutic studies.

• Experimental Control and Reversibility: Using recombinant proteins (CNTF and CNTFRα) allows for precise dosing and timing, as opposed to gene therapy approaches, and enables researchers to study both acute and reversible effects of CNTF signaling.

• Mechanistic Studies: Recombinant CNTFRα is valuable for dissecting the specific roles of CNTF signaling in cell differentiation, survival, and gene expression, as it allows researchers to selectively activate or inhibit the pathway in vitro and in vivo.

Typical applications include:

• Neuroprotection studies in retinal degeneration, ALS, and other neurodegenerative models.
• Investigation of CNTF’s effects on photoreceptor differentiation and survival.
• Elucidation of downstream signaling pathways and gene expression changes in response to CNTF.
• Testing therapeutic strategies that combine CNTF and CNTFRα for enhanced efficacy in preclinical models.

Summary:
Recombinant Rat CNTF Rα is a critical reagent for enabling and modulating CNTF signaling in research, especially when studying neuroprotection, retinal degeneration, and neuronal differentiation, or when working with cell types lacking endogenous receptor expression.

Recombinant Rat CNTF Rα can be used as a standard for quantification or calibration in ELISA assays, but only if the ELISA kit or protocol is specifically designed and validated for detecting CNTF Rα (the receptor alpha subunit), not CNTF itself.

Here are the key considerations:

• Target Specificity: Most ELISA kits for CNTF are designed to detect the cytokine CNTF, not its receptor CNTF Rα. Using recombinant CNTF Rα as a standard in a CNTF-specific ELISA will not yield accurate quantification because the antibodies in the kit are unlikely to recognize the receptor.

• Kit Compatibility: If you are using an ELISA kit intended for CNTF Rα detection (such as those listed for Rat CNTF R alpha ELISA), then recombinant Rat CNTF Rα is appropriate as a standard. Always check the kit's datasheet to confirm the recommended standard and cross-reactivity.

• Validation: Even if the kit is compatible, it is important to validate that the recombinant protein behaves similarly to the native protein in your assay system (e.g., similar binding kinetics, lack of interference).

• Standard Preparation: Follow the manufacturer’s instructions for preparing the standard curve, including the recommended diluent and concentration range.

In summary, yes, you can use recombinant Rat CNTF Rα as a standard, but only in ELISAs specifically designed for CNTF Rα detection, not for CNTF. Always verify compatibility with your specific kit and assay conditions.

Recombinant Rat Ciliary Neurotrophic Factor (CNTF) and its receptor system (including CNTFRα) have been validated in published research for several key applications, particularly in the context of neuroprotection and retinal disease. Here are the main applications supported by the literature:

1. Neuroprotection in Retinal Degeneration

• Photoreceptor Survival: Recombinant rat CNTF has been shown to exert a long-term photoreceptor preservative effect in animal models of retinal degeneration, such as the Royal College of Surgeons (RCS) rat. A single subretinal injection of recombinant CNTF provided protection up to 36 days post-injection .
• Retinal Ganglion Cell (RGC) Survival: CNTF treatment improves the survival of retinal ganglion cells and promotes axonal regeneration in models of optic nerve injury, including optic nerve crush or transection .
• Gene Therapy: Adeno-associated virus (AAV)-mediated delivery of CNTF has demonstrated protective effects against photoreceptor loss in retinal diseases, including retinitis pigmentosa (RP) .

2. Neuroprotection in Neurodegenerative Diseases

• Amyotrophic Lateral Sclerosis (ALS): In rat models of ALS, CNTF significantly reduced motor neuron destruction, improved motor function, and extended survival .
• Huntington’s Disease: CNTF has shown neuroprotective effects in both primate and mouse models of Huntington’s disease .

3. Mechanistic Studies of CNTF Signaling

• Receptor Binding and Activity: Recombinant rat CNTF has been used to study the binding and activation of the CNTF receptor complex, which includes CNTFRα, gp130, and LIFRβ. Rat CNTF exhibits higher biological activity and receptor binding affinity compared to human CNTF in certain assays .
• Metabolic Effects: CNTF signaling has been shown to influence retinal metabolism, leading to increased aerobic glycolysis and enhanced anabolism, which contributes to neuronal viability in degenerating retinas .

4. Cell Culture and In Vitro Studies

• Neuronal Survival and Neurite Outgrowth: Recombinant rat CNTF supports the survival and neurite outgrowth of embryonic chick ciliary neurons in culture, demonstrating its utility in in vitro neuroprotection studies .
• Glial Cell Activation: CNTF stimulates astrocytes to secrete FGF-2 and microglia to secrete GDNF, highlighting its role in modulating glial cell function .

5. Pharmacological and Therapeutic Development

• Modified CNTF Variants: Studies have used recombinant rat CNTF as a benchmark to develop modified human CNTF variants with enhanced biological activity and therapeutic index, which are being explored for clinical applications in neurological and retinal diseases .

6. Metabolic and Behavioral Effects

• Appetite and Body Weight Regulation: CNTF has been shown to reduce appetite and body weight in rodents and humans, indicating its broader physiological roles beyond neuroprotection .

Summary

Recombinant Rat CNTF has been validated in published research for:

• Neuroprotection in retinal and neurodegenerative diseases.
• Mechanistic studies of CNTF receptor signaling and metabolic effects.
• In vitro neuronal survival and glial cell activation.
• Therapeutic development of modified CNTF variants.
• Metabolic and behavioral studies.

These applications highlight the versatility and importance of recombinant rat CNTF in both basic research and translational studies.

To reconstitute and prepare Recombinant Rat CNTF Rα protein for cell culture experiments, follow these best-practice steps based on general recombinant protein handling protocols and specific guidance for CNTF Rα:

1. Centrifuge the Vial
   Briefly centrifuge the lyophilized protein vial before opening to ensure all material is collected at the bottom and to minimize loss.

2. Choose the Appropriate Reconstitution Buffer

   • For most recombinant proteins, sterile phosphate-buffered saline (PBS, pH 7.4) or sterile distilled water is suitable unless otherwise specified in the product datasheet or Certificate of Analysis (CoA).
   • For CNTF Rα, PBS (pH 7.4) is commonly used, sometimes with added glycerol for stability.

3. Reconstitution Concentration

   • Typical reconstitution concentrations are 0.1–1.0 mg/mL.
   • For example, to achieve 0.5 mg/mL, add 200 μL buffer to 100 μg lyophilized protein.

4. Dissolve the Protein

   • Gently mix (do not vortex) and allow the protein to dissolve at room temperature for 15–30 minutes with gentle agitation.
   • If particulates remain, continue gentle mixing at room temperature or at 4°C for up to several hours.

5. Aliquot and Storage

   • Prepare small aliquots to avoid repeated freeze-thaw cycles, which can reduce protein activity.
   • For short-term storage (up to 1 month), keep reconstituted protein at 2–8°C under sterile conditions.
   • For long-term storage, add a carrier protein (e.g., 0.1% BSA or 10% glycerol) to prevent adsorption and degradation, then store at –20°C to –80°C.

6. Working Solution Preparation

   • Before adding to cell culture, dilute the reconstituted stock to the desired working concentration using cell culture medium or buffer containing a carrier protein to minimize loss by adsorption.
   • Avoid using animal-derived carriers (e.g., BSA, FBS) if your experiment requires serum-free conditions; consider trehalose as an alternative stabilizer.

7. Handling Precautions

   • Avoid vigorous shaking or foaming, which can denature the protein.
   • Use sterile technique throughout to prevent contamination.

Example Protocol for Recombinant Rat CNTF Rα:

• Centrifuge the vial.
• Add sterile PBS (pH 7.4) to achieve 0.5 mg/mL.
• Gently mix and incubate at room temperature for 15–30 minutes.
• Aliquot and store at –20°C to –80°C with 10% glycerol if storing long-term.
• Thaw aliquots on ice and dilute to working concentration in cell culture medium immediately before use.

Note: Always consult the specific product datasheet or CoA for any unique requirements for your batch of recombinant protein, as formulations and recommended buffers may vary.