Recombinant Rat CXCL7

Recombinant rat CXCL7 offers several compelling advantages for research applications, particularly in studies examining chemokine signaling, immune responses, and disease mechanisms.

Biological Activity and Functional Relevance

Recombinant rat CXCL7 (also known as NAP-2) is a functional CXC chemokine that retains its biological activity for experimental use. This protein effectively chemoattracts and activates neutrophils, making it valuable for investigating neutrophil recruitment and activation pathways. The protein can be produced with high purity (>97% by SDS-PAGE) and low endotoxin levels (<0.1 EU/µg), ensuring reliable and reproducible experimental results.

Research Applications

Inflammatory Disease Models: Recombinant rat CXCL7 is particularly useful for studying inflammatory conditions. Research demonstrates that CXCL7 plays a significant role in rheumatoid arthritis by enhancing RANKL-induced osteoclastogenesis through ERK/NFATc1 signaling pathways. Using recombinant CXCL7 allows you to investigate these mechanisms in vitro and establish dose-response relationships in cellular systems.

Receptor Signaling Studies: The protein binds to CXCR1 and CXCR2 receptors, enabling investigation of chemokine receptor signaling in multiple contexts. You can use recombinant rat CXCL7 to study receptor activation, downstream signaling cascades, and cellular responses in transfected cell systems or primary cell cultures.

Tissue Regeneration Research: Recombinant CXCL7 has demonstrated utility in studying tissue repair mechanisms, as platelet-derived CXCL7 promotes skeletal muscle regeneration by recruiting neutrophils to injured tissues. This makes it valuable for regenerative medicine research applications.

Comparative and Cross-Species Studies: Using rat-derived recombinant protein allows for species-specific investigations and validation of findings across different mammalian models, which is particularly important when planning subsequent in vivo studies in rodent disease models.

The combination of high purity, confirmed biological activity, and low endotoxin contamination makes recombinant rat CXCL7 a reliable reagent for mechanistic studies of chemokine function in inflammation, immune cell recruitment, and tissue homeostasis.

Yes, recombinant Rat CXCL7 can be used as a standard for quantification or calibration in ELISA assays, provided it is of high purity and its concentration is accurately determined. This is a common practice in quantitative ELISA protocols for cytokines and chemokines, including CXCL7.

Essential context and supporting details:

• ELISA Standard Preparation: Quantitative ELISA assays require a standard curve generated from known concentrations of the target analyte. Recombinant proteins, such as recombinant Rat CXCL7, are routinely used for this purpose when purified native protein is unavailable.
• Purity and Quantification: The recombinant CXCL7 should be highly purified and its concentration verified, typically by absorbance at 280 nm or another protein quantification method. Impurities or inaccurate concentration can compromise the standard curve and assay accuracy.
• Reconstitution and Storage: Follow manufacturer or supplier instructions for reconstitution and storage. For example, some protocols recommend storing reconstituted recombinant CXCL7 at −20°C or −80°C and using aliquots to avoid repeated freeze-thaw cycles.
• Matrix Effects: When preparing standards, dilute recombinant CXCL7 in the same buffer or matrix as your samples (e.g., assay diluent, serum, plasma) to minimize matrix effects and ensure comparable binding and detection.
• Validation: It is good practice to validate that the recombinant standard behaves similarly to endogenous CXCL7 in your assay system. This can be confirmed by spike-and-recovery experiments and parallelism testing.

Additional relevant information:

• ELISA Kits: Many commercial rat CXCL7 ELISA kits use recombinant rat CXCL7 as the standard for calibration, confirming its suitability for this application.
• Carrier Proteins: Some recombinant proteins are supplied with carrier proteins (e.g., BSA) to improve stability. Carrier-free preparations are preferred for ELISA standards to avoid interference, unless otherwise validated.
• Documentation: Always refer to the ELISA kit or recombinant protein datasheet for specific instructions regarding standard preparation, dilution, and storage.

In summary, recombinant Rat CXCL7 is appropriate for use as a standard in ELISA quantification, provided best practices for purity, quantification, and validation are followed.

Research Applications and Validation

Recombinant rat CXCL7 has been validated across several important research applications, primarily centered on its role as a chemokine in inflammatory and immunological processes.

Cellular Chemotaxis and Activation Studies

The recombinant protein has been validated for assessing neutrophil chemoattraction and activation. Specifically, the biological activity of recombinant rat CXCL7 has been determined through its ability to chemoattract cells expressing CXCR2 receptors, such as transfected mouse BaF/3 cells. This application is fundamental for studying the chemokine's role in neutrophil recruitment and immune cell migration.

Signaling Pathway Analysis

Recombinant CXCL7 has been employed in Western blotting studies to investigate downstream signaling mechanisms. Research has demonstrated that CXCL7 enhances phosphorylation of ERK1/2 and promotes NFATc1 expression in CD14+ monocytes, particularly in the context of RANKL-induced osteoclastogenesis. These applications validate the protein's utility in examining intracellular signaling cascades relevant to bone resorption and inflammatory arthritis.

Immunoassay Applications

The recombinant protein serves as a standard and detection target in enzyme-linked immunosorbent assays (ELISA) and sandwich ELISA formats. These applications are critical for quantifying endogenous CXCL7 levels in biological samples and validating antibody specificity and cross-reactivity profiles.

Functional Characterization

Recombinant rat CXCL7 has been validated for stimulating various cellular processes including mitogenesis, extracellular matrix synthesis, glucose metabolism, and plasminogen activator synthesis. These applications establish the protein's multifaceted roles in cellular physiology and metabolic regulation.

The high purity (>97% by SDS-PAGE) and low endotoxin levels (<0.1 EU/µg) of recombinant rat CXCL7 make it suitable for these sensitive research applications requiring reliable biological activity and minimal confounding factors.

To reconstitute and prepare Recombinant Rat CXCL7 protein for cell culture experiments, follow these best-practice steps based on current protocols and manufacturer recommendations:

1. Centrifuge the vial:
Before opening, briefly centrifuge the lyophilized protein vial to ensure all material is collected at the bottom.

2. Choose the correct reconstitution buffer:

• If the protein is carrier-free (no BSA): Reconstitute in sterile PBS (phosphate-buffered saline), typically at a concentration of 100 μg/mL.
• If the protein contains a carrier protein (e.g., BSA): Reconstitute in sterile PBS containing at least 0.1% human or bovine serum albumin (BSA), usually at 25 μg/mL.

3. Reconstitution procedure:

• Add the appropriate volume of buffer directly to the vial to achieve the desired concentration.
• Gently swirl or invert the vial to dissolve the protein. Avoid vigorous vortexing, which can denature the protein.
• Allow the solution to sit at room temperature for 10–30 minutes to ensure complete dissolution.

4. Aliquot and storage:

• After reconstitution, aliquot the solution into small volumes to avoid repeated freeze-thaw cycles, which can degrade the protein.
• Store aliquots at –20°C to –70°C for long-term storage. For short-term use (up to one week), store at 2–8°C.
• Avoid multiple freeze-thaw cycles by using single-use aliquots.

5. Preparation for cell culture:

• Before adding to cell cultures, dilute the reconstituted protein to the desired working concentration using cell culture medium or PBS with 0.1% BSA to minimize adsorption to plastic and maintain protein stability.
• Filter sterilize the final working solution if sterility is required and the buffer composition allows.

6. General notes:

• Always consult the specific product datasheet for any lot-specific instructions, as buffer composition and recommended concentrations may vary.
• If using in sensitive assays or with cells that may react to BSA, use the carrier-free formulation and supplement with BSA only if needed for stability.

Summary Table:

Key technical tips:

• Use sterile technique throughout to prevent contamination.
• Avoid repeated freeze-thaw cycles.
• If unsure about buffer compatibility with your cells, perform a small-scale pilot test.

These steps will ensure optimal solubility, stability, and biological activity of recombinant rat CXCL7 for cell culture applications.