Recombinant Rat IL-5

Recombinant rat IL-5 is a valuable tool for research applications due to its well-characterized biological activities and versatile experimental utility. This cytokine offers several compelling reasons for inclusion in your research program.

Biological Activity and Functional Characterization

Recombinant rat IL-5 demonstrates robust and reproducible biological activities in vitro and in vivo. The protein promotes proliferation of specific cell lines and induces terminal differentiation and proliferation of progenitor cells to mature eosinophils in rats. This makes it particularly useful for studying eosinophil biology and development. The protein exhibits dose-dependent effects, with effective concentrations in the range of 0.5–2.5 ng/mL for inducing proliferation in certain cell systems.

Dual Cellular Targets

One of the key advantages of recombinant rat IL-5 is its ability to act on multiple cell types. Through binding to the IL-5 receptor, the protein stimulates B cell growth and increases immunoglobulin secretion while simultaneously serving as a key mediator in eosinophil activation. This dual functionality allows you to investigate both adaptive immune responses and innate immune cell development within a single experimental framework.

Signaling Pathway Applications

Recombinant rat IL-5 activates multiple intracellular signaling cascades that are relevant for mechanistic studies. The protein triggers JAK2/STAT pathway activation, which regulates genes involved in eosinophil proliferation such as pim-1 and cyclin D3. Additionally, IL-5 activates class IA PI3K, ERK, and PKCδ pathways, enabling investigation of adhesion, degranulation, and cytokine production mechanisms. These diverse signaling routes make the protein suitable for comprehensive pathway analysis studies.

Validated Experimental Applications

Recombinant rat IL-5 has been validated for use in multiple experimental techniques including ELISA and functional assays. The availability of different formulations, such as His-tagged variants and carrier-free preparations, provides flexibility in experimental design and downstream applications. This standardization facilitates reproducibility and comparison across studies.

Research Relevance

The protein is particularly valuable for investigating allergic responses, eosinophilic disorders, and immune regulation, as IL-5 plays central roles in eosinophil differentiation, maturation, migration, and survival. If your research focuses on these areas, recombinant rat IL-5 provides a physiologically relevant tool for mechanistic investigation.

Yes, recombinant rat IL-5 can be used as a standard for quantification or calibration in ELISA assays, provided it is of high purity and properly validated for your specific assay system. Recombinant cytokines are commonly used as standards in quantitative ELISA protocols to generate standard curves, which allow for the accurate determination of analyte concentrations in biological samples.

Key considerations for using recombinant rat IL-5 as an ELISA standard:

• Purity and Activity: The recombinant IL-5 should be highly pure (typically >95% by SDS-PAGE/HPLC) and biologically active, as impurities or inactive protein can affect quantification accuracy.
• Validation: Confirm that the recombinant IL-5 is recognized by the antibodies used in your ELISA kit. Most commercial rat IL-5 ELISA kits are validated to detect recombinant rat IL-5, and their protocols often use recombinant protein as the reference standard for calibration.
• Preparation: Follow the kit’s instructions for reconstitution and dilution of the standard. Proper handling ensures reproducibility and accuracy of the standard curve.
• Matrix Effects: If you are spiking recombinant IL-5 into biological matrices (e.g., serum or plasma), ensure that recovery and linearity have been validated, as matrix components can affect detection.

Protocol context:

• ELISA kits for rat IL-5 typically use a sandwich format, where the standard curve is generated using serial dilutions of recombinant rat IL-5.
• The standard curve allows interpolation of sample concentrations based on their optical density (OD) readings at 450 nm.
• Recombinant rat IL-5 is specifically mentioned as suitable for standard curve generation in validated ELISA protocols.

Best practices:

• Always use the same batch of recombinant IL-5 for calibration to minimize variability.
• Store aliquots at recommended conditions to preserve protein integrity.
• Include appropriate controls (positive and negative) to ensure assay reliability.

In summary: Recombinant rat IL-5 is widely accepted and routinely used as a standard for ELISA quantification, as long as it is compatible with your assay’s antibodies and protocol. Always consult your specific ELISA kit documentation for any additional requirements or recommendations.

Recombinant Rat IL-5 has been validated in published research for several key applications, primarily in studies of immune cell differentiation, function, and disease modeling.

Validated Applications in Published Research:

• Functional Assays: Recombinant rat IL-5 has been used to demonstrate its role as a B cell growth factor, an eosinophil differentiation factor, and an eosinophil survival-enhancing factor. These activities are typically validated using in vitro cell culture systems where IL-5 is added to assess its effects on B cell proliferation, eosinophil maturation, and survival.

• Cell Culture Supplementation: It is commonly used as a cytokine supplement in primary cell cultures or cell lines to induce eosinophil proliferation and differentiation from bone marrow progenitors, especially in models of allergic disease or helminth infection.

• ELISA Standard: Recombinant rat IL-5 is validated as a standard in ELISA assays for quantifying IL-5 levels in biological samples. This enables accurate measurement of endogenous IL-5 in rat serum, plasma, or cell culture supernatants.

• Disease Modeling: In vivo and ex vivo studies use recombinant rat IL-5 to model allergic inflammation, asthma, and parasitic infections, where it induces eosinophilia and modulates immune responses.

Supporting Details:

• IL-5’s biological activities have been confirmed in published studies, showing its ability to stimulate B cell growth, enhance immunoglobulin secretion, and promote eosinophil activation and survival.
• It is also used to study the differentiation of basophils and its role in cell-mediated immunity against parasites and certain tumors.
• The protein is highly conserved across species, supporting its use in comparative immunology studies.

Summary Table of Validated Applications

These applications are well-supported in the literature and are considered standard for recombinant rat IL-5 in immunological research.

To reconstitute and prepare Recombinant Rat IL-5 protein for cell culture experiments, follow these best-practice steps:

1. Reconstitution

• Centrifuge the vial briefly (e.g., 10,000 rpm for 1 minute) to collect all lyophilized powder at the bottom.
• Add sterile water to the vial to achieve your desired stock concentration (commonly 100 μg/mL or as specified by your protocol).
• Allow the vial to stand at room temperature for 10–30 minutes to fully dissolve the protein. Gently swirl or invert; avoid vigorous shaking or vortexing to prevent foaming and denaturation.
• If the protein does not dissolve easily, gentle pipetting or low-speed vortexing may be used, but avoid introducing bubbles.

2. Preparation for Cell Culture

• Once fully dissolved, dilute the stock solution to your working concentration using sterile cell culture-grade buffer (e.g., PBS or cell culture medium). For most applications, include a carrier protein such as 0.1% BSA or 10% FBS to stabilize the protein and minimize adsorption to plastic.
• Filter-sterilize the final solution if sterility is required and the protein is not already in a sterile buffer.

3. Storage

• Aliquot the reconstituted stock to avoid repeated freeze-thaw cycles.
• Store aliquots at –20°C to –80°C for long-term use; at 2–8°C for up to one week.
• Avoid repeated freeze-thaw cycles, as this can reduce protein activity.

4. Additional Tips

• Always bring all reagents to room temperature before use in cell culture experiments.
• Avoid foaming during all handling steps, as this can denature the protein.
• Refer to the specific product datasheet for any unique instructions regarding buffer composition or reconstitution volume, as some preparations may contain stabilizers like trehalose or mannitol.

Example Protocol (for a 100 μg vial):

1. Spin down vial briefly.2. Add 1 mL sterile water to achieve 100 μg/mL.3. Gently swirl and let stand at room temperature for 15–30 minutes.4. Aliquot and store at –80°C.5. For cell culture, dilute to working concentration in medium with 0.1% BSA.

Summary of Key Points:

• Use sterile water for initial reconstitution.
• Avoid vigorous agitation and foaming.
• Dilute with carrier protein for stability.
• Aliquot and store at low temperatures to preserve activity.

These steps will ensure optimal activity and reproducibility of recombinant rat IL-5 in cell culture experiments.