Recombinant Rat CNTF

Scientific Rationale for Recombinant Rat CNTF in Research

Recombinant rat CNTF (ciliary neurotrophic factor) is a valuable tool for neuroscience research due to its well-characterized neuroprotective and neurotrophic properties. This cytokine offers multiple advantages for investigating neuronal survival, differentiation, and degeneration mechanisms.

Neuroprotective Capabilities

CNTF functions as a potent survival factor for various neuronal cell types, making it particularly useful for studying neuroprotection mechanisms. The protein demonstrates robust protective effects against retinal ganglion cell death under oxidative stress conditions, with studies showing enhanced viability and β3-tubulin expression in treated cells. This makes it especially valuable for research involving degenerative diseases and stress-induced neuronal damage.

In retinal degeneration models, recombinant rat CNTF has proven effective at preserving photoreceptor morphology and function. The protein's ability to prevent axon degeneration following axotomy provides another important research application for studying axonal injury and regeneration.

Mechanistic Insights

Research utilizing recombinant rat CNTF has revealed important signaling pathways underlying neuroprotection. Studies demonstrate that CNTF activates multiple intracellular cascades including the PI3K/AKT pathway (associated with cell viability), the JAK/STAT pathway (enhancing RGC viability), and the MAPK/ERK pathway (linked to cytoskeletal protein expression). Additionally, transcriptome analysis reveals that CNTF upregulates proteolysis inhibitors, which may prevent cellular and extracellular matrix degradation.

Practical Research Applications

Recombinant rat CNTF is validated for multiple experimental techniques including functional assays and ELISA applications. The protein's biological activity can be confirmed through phosphorylation of STAT3 in cell-based assays, providing a reliable readout for bioactivity validation. Its use in both in vitro cell culture systems and in vivo animal models makes it versatile for comprehensive mechanistic studies spanning from molecular signaling to whole-organism efficacy evaluation.

Yes, recombinant rat CNTF can be used as a standard for quantification or calibration in ELISA assays, provided it is compatible with your assay system and matches the native protein recognized by your antibodies. This is a common practice in quantitative ELISA protocols for cytokines and growth factors, including CNTF.

Key considerations and supporting details:

• Recombinant CNTF is routinely used as a standard in commercial rat CNTF ELISA kits. These kits include recombinant rat CNTF as the reference standard to generate a standard curve for quantification of CNTF in biological samples. The standard curve is essential for determining the concentration of CNTF in unknown samples by comparing their signal to that of known concentrations of the recombinant standard.

• Standard preparation and dilution: The recombinant CNTF standard is typically reconstituted and serially diluted in the provided standard diluent to cover the assay’s quantification range (e.g., 8–2000 pg/mL). It is important to follow the kit or assay protocol for dilution and storage to ensure accuracy and reproducibility.

• Assay compatibility: The recombinant standard should be of the same species and sequence as the target analyte in your samples. Most rat CNTF ELISA kits are validated for both natural and recombinant rat CNTF, ensuring that the antibodies used in the assay recognize the recombinant form equivalently to the endogenous protein.

• Carrier protein considerations: Some recombinant proteins are supplied with carrier proteins (e.g., BSA) to enhance stability. For ELISA standards, both carrier-free and carrier-containing forms can be used, but carrier-free is preferred if you want to avoid potential interference or background.

• Validation and controls: It is good practice to validate that your recombinant CNTF standard produces a parallel standard curve to the endogenous CNTF in your sample matrix. This ensures that matrix effects do not alter quantification accuracy. Internal controls and spike-recovery experiments can further confirm assay performance.

• Exceptions: Some ELISA kits may specify that they are not validated for detection of recombinant proteins from certain sources or with specific tags. Always check the assay documentation to confirm compatibility.

Summary Table: Use of Recombinant Rat CNTF as ELISA Standard

In conclusion, recombinant rat CNTF is appropriate for use as a standard in ELISA quantification, provided it is compatible with your assay’s antibodies and protocol. Always verify with your specific assay documentation and perform appropriate controls to ensure accurate quantification.

Recombinant Rat CNTF has been validated for several key applications in published research:

Functional and Cell-Based Assays

Recombinant Rat CNTF demonstrates robust activity in functional assays measuring neuronal responses. The protein has been validated for its ability to support neuronal survival and stimulate neurite outgrowth in dissociated chick embryonic dorsal root ganglia (DRG) neurons. Additionally, the protein induces proliferation of TF-1 human erythroleukemic cells in a dose-dependent manner, with an ED50 typically ranging from 3-18 ng/mL. The protein also enhances cell proliferation through interaction with its receptor complex, an effect that can be neutralized by specific antibodies.

Immunological Applications

The protein has been validated for use in Western blot analysis, where it can be detected using specific antibodies against the CNTF receptor alpha component. Immunohistochemistry applications have also been established, with the protein successfully used in perfusion-fixed frozen sections of rat brain tissue, particularly in cortical regions.

Blocking and Neutralization Studies

Recombinant Rat CNTF serves as a critical reagent in neutralization assays, where antibodies against CNTF receptor components can be titrated to block CNTF-mediated biological effects. These assays typically employ the protein at concentrations around 10 ng/mL in combination with receptor components.

ELISA Applications

The protein has been validated for enzyme-linked immunosorbent assay (ELISA) applications, enabling quantitative detection and measurement of CNTF in various experimental contexts.

In Vivo Efficacy Studies

Recombinant Rat CNTF has demonstrated significant neuroprotective effects in animal models of retinal degeneration. Single subretinal injections showed long-term photoreceptor preservation effects extending up to 36 days in Royal College of Surgeons (RCS) rat models. Intravitreal administration of the recombinant protein has been evaluated for its ability to penetrate retinal tissue and provide neuroprotection against photoreceptor degeneration.

Human recombinant CNTF, which shares structural and functional homology with the rat ortholog, has been extensively studied in clinical contexts for treating hereditary and age-related retinal degenerative diseases, demonstrating metabolic and neuroprotective mechanisms in degenerating retinal tissue.

To reconstitute and prepare Recombinant Rat CNTF protein for cell culture experiments, first check the product datasheet for any supplier-specific instructions. In the absence of unique requirements, follow these general best practices:

Reconstitution Steps:

• Centrifuge the vial briefly to collect all lyophilized protein at the bottom before opening.
• Add sterile water or buffer: Most protocols recommend reconstituting in sterile distilled water or PBS. Some sources suggest using 0.4% NaHCO₃ (pH 8–9) for improved solubility, especially if the protein is difficult to dissolve.
• Concentration: Reconstitute to a final concentration of 0.1–1.0 mg/mL (100–1000 µg/mL). For example, add 100 µL to 100 µg of protein for a 1 mg/mL solution.
• Dissolve gently: Do not vortex. Mix by gentle pipetting or swirling. Allow the protein to dissolve at room temperature for 15–30 minutes. If undissolved material remains, gently mix for up to 2 hours.
• Carrier protein: For long-term storage or to prevent adsorption, dilute further in buffer containing a carrier protein such as 0.1% BSA or 5% trehalose, especially for working aliquots.

Storage:

• Short-term: Store reconstituted protein at 2–8°C for up to 1 week.
• Long-term: Aliquot and store at –20°C or –80°C. Avoid repeated freeze-thaw cycles.
• Lyophilized protein: Store desiccated at –20°C or below.

Preparation for Cell Culture:

• Dilution: Before adding to cell cultures, dilute the reconstituted CNTF to the desired working concentration using sterile cell culture medium or buffer. Include a carrier protein if possible to minimize loss from adsorption.
• Sterility: Always use sterile technique and solutions. If sterility is uncertain, filter the final solution through a 0.22 µm filter.

Summary Table:

Key Notes:

• Do not vortex to avoid protein denaturation.
• Avoid repeated freeze-thaw cycles to maintain activity.
• Check for visible solubility; if insoluble, adjust pH or buffer as needed.

These guidelines are broadly applicable for recombinant rat CNTF protein from most sources. Always consult the specific product datasheet for any unique requirements.