Recombinant Human gp130

Using Recombinant Human gp130 in research applications is valuable for dissecting and modulating cytokine signaling, particularly within the interleukin-6 (IL-6) family, and for developing therapeutic strategies targeting inflammation, autoimmunity, and cancer.

Key scientific reasons to use recombinant human gp130 include:

• Deciphering Cytokine Signaling Pathways: gp130 is the common signal-transducing receptor subunit for the IL-6 family of cytokines (including IL-6, IL-11, LIF, OSM, CT-1, CLCF1, and CNTF). Recombinant gp130 enables controlled in vitro studies of these pathways, allowing researchers to analyze downstream effects such as JAK/STAT, ERK, and PI3K activation.

• Blocking Pathological Cytokine Activity: The soluble form of gp130 (sgp130) acts as a natural inhibitor of IL-6 trans-signaling by sequestering the IL-6/sIL-6R complex, thereby preventing excessive or pathological activation of gp130-mediated pathways. Recombinant sgp130 is used to specifically block disease-relevant trans-signaling, which is implicated in chronic inflammation, autoimmune diseases, and certain cancers.

• Therapeutic and Functional Studies: Recombinant gp130 is instrumental in preclinical models for evaluating the efficacy of blocking IL-6 family cytokine signaling in conditions such as inflammatory arthritis, inflammatory bowel disease, and cancer. It is also used to test the effects of novel inhibitors (e.g., nanobodies or antibodies) targeting gp130 or its ligand complexes.

• Bioassays and Mechanistic Investigations: Recombinant gp130 is used in cell-based assays to:

  • Assess cytokine-induced cell proliferation, differentiation, or survival.
  • Measure downstream phosphorylation events (e.g., STAT3, ERK).
  • Evaluate the inhibitory potential of candidate molecules on cytokine signaling.

• Disease Modeling: By modulating gp130 activity in vitro or in vivo, researchers can model disease states driven by aberrant cytokine signaling, such as chronic inflammation, fibrosis, or tumor progression, and test potential therapeutic interventions.

• Tool for Receptor-Ligand Interaction Studies: Recombinant gp130 is essential for structural and biochemical studies of cytokine-receptor complexes, helping to elucidate mechanisms of signal transduction and inform drug design.

In summary, recombinant human gp130 is a versatile reagent for:

• Dissecting IL-6 family cytokine signaling mechanisms.
• Blocking or mimicking gp130-mediated pathways in disease models.
• Screening and characterizing therapeutic candidates targeting cytokine signaling.
• Advancing understanding of immune regulation, hematopoiesis, tissue repair, and oncogenesis.

When using recombinant gp130, consider the specific isoform (membrane-bound vs. soluble), experimental context, and desired readouts to ensure relevance and interpretability of results.

You can use recombinant human gp130 as a standard for quantification or calibration in your ELISA assays, provided that the recombinant protein is of high purity, its concentration is accurately known, and it is compatible with the antibodies and detection system used in your specific ELISA format.

Key considerations and supporting details:

• ELISA Standards: Commercial ELISA kits for human gp130 typically use recombinant human gp130 as the standard to generate the calibration curve. The standard curve is essential for quantifying gp130 concentrations in unknown samples by comparing their optical density (OD) values to those of known concentrations of the standard.
• Compatibility: The recombinant gp130 standard must be recognized by the capture and detection antibodies in your assay. Most sandwich ELISAs are designed to detect both natural and recombinant forms, but you should confirm this in your assay documentation or by running a pilot experiment.
• Standard Preparation: The recombinant gp130 should be reconstituted and diluted according to best practices, ensuring accurate concentration and stability. Use the same buffer as your samples or as recommended in your ELISA protocol to minimize matrix effects.
• Documentation: Some recombinant gp130 proteins are specifically labeled as suitable for use as ELISA standards, often provided with a certificate of analysis and instructions for use in standard curve preparation.
• Validation: If you are developing your own ELISA or using a non-kit format, validate the recombinant standard by assessing its linearity, recovery, and parallelism with endogenous gp130 in your sample matrix.

Limitations:

• The recombinant standard should be of high purity and well-characterized. Impurities or incorrect folding may affect antibody recognition.
• If your ELISA is designed for a specific isoform or post-translational modification, ensure the recombinant standard matches the target analyte.

Summary:
Recombinant human gp130 is widely used as a standard in ELISA assays for quantification and calibration, provided it is compatible with your assay system and properly validated. Always follow best practices for standard preparation and validation to ensure accurate results.

Recombinant Human gp130 has been validated in published research for several key applications, primarily in bioassays, ELISA (capture), neutralization studies, and as a control in immunocytochemistry and related protocols.

Validated Applications in Published Research:

• Bioassays:
  Recombinant human gp130 is widely used in cell-based bioassays to study cytokine signaling, particularly for the IL-6 family of cytokines (including IL-6, IL-11, LIF, OSM, CNTF, CT-1, CLCF1, and IL-27). These assays often assess the activation or inhibition of downstream signaling pathways such as JAK/STAT, ERK, and PI3K in various cell types.

• ELISA (Capture):
  It has been validated as a capture reagent in ELISA to detect ligands or complexes involving gp130, such as soluble IL-6 receptor/IL-6 complexes or oncostatin M.

• Neutralization:
  Recombinant gp130 has been used in neutralization assays to block cytokine signaling, particularly to inhibit IL-6 trans-signaling or OSM/CNTF activity in vitro. This is relevant for dissecting the role of gp130-mediated pathways in inflammation and cancer models.

• Control:
  It serves as a control protein in immunocytochemical detection and RT-PCR studies, for example, to confirm the specificity of antibodies or primers targeting gp130 or its ligands.

Additional Research Uses:

• Structural and Biochemical Studies:
  Recombinant gp130 is used for structural biology (e.g., crystallography, cryo-EM) to elucidate cytokine-receptor complex formation and for pull-down assays to map binding epitopes.

• Functional Characterization:
  It is employed to characterize the biological activity of cytokines, study receptor-ligand interactions, and investigate the effects of soluble gp130 isoforms in disease models such as inflammatory arthritis, cancer, and diabetes.

• Therapeutic Target Validation:
  Recombinant gp130 is used in preclinical studies to validate its role as a therapeutic target in models of inflammation, fibrosis, and cancer, often by blocking or mimicking its signaling.

Summary Table of Validated Applications

Key Points:

• The most common and well-validated application is in bioassays for cytokine signaling research.
• It is also validated for use in ELISA as a capture reagent and in neutralization assays.
• Additional uses include structural biology, control experiments, and therapeutic target validation in preclinical models.

If you need protocol-specific details or further information on a particular application, please specify the context or research focus.

Reconstitution Protocol

Recombinant human gp130 proteins are typically supplied in lyophilized form and require proper reconstitution before use in cell culture experiments. The reconstitution process is straightforward but requires attention to specific details to maintain protein integrity and biological activity.

Initial Preparation Steps

Begin by centrifuging the vial to concentrate the lyophilized powder at the bottom of the tube. This ensures complete dissolution when the reconstitution buffer is added. Allow adequate time for the protein to fully dissolve—typically 15-30 minutes at room temperature with gentle agitation. Avoid vigorous shaking, as this can cause foaming and potentially denature the protein.

Reconstitution Buffers and Concentrations

Standard Reconstitution

For most applications, reconstitute the protein at 100 μg/mL in sterile PBS (phosphate-buffered saline). This concentration is suitable for cell culture work and provides a practical working stock.

Carrier Protein Considerations

The choice between carrier-containing and carrier-free formulations is important for your specific application:

• With carrier protein (BSA): Reconstitute at 100 μg/mL in sterile PBS containing at least 0.1% human or bovine serum albumin. This formulation enhances protein stability, increases shelf-life, and allows storage at more dilute concentrations. Use this version for cell or tissue culture applications.

• Carrier-free formulation: Reconstitute at 100 μg/mL in sterile PBS alone. Some carrier-free versions may be lyophilized with trehalose as a stabilizer. This formulation is recommended when the presence of BSA could interfere with your experimental results.

Storage and Stability

Temperature Management

Store the reconstituted protein immediately upon receipt at the temperature specified in your product documentation. Use a manual defrost freezer and avoid repeated freeze-thaw cycles, as these can compromise protein activity and stability.

Long-term Storage

Lyophilized protein is typically stable for 6-12 months when stored desiccated at -20°C to -70°C. After reconstitution, follow the stability guidelines provided with your specific product, as these vary depending on formulation and additives.

Quality Considerations for Cell Culture

When preparing gp130 for cell culture experiments, verify that your protein meets appropriate quality standards. Typical specifications include purity >90% by SDS-PAGE analysis and endotoxin levels <0.01 EU/μg, which are critical for cell culture applications where endotoxin contamination could confound results.

The biological activity of gp130 can be confirmed through functional assays, such as its ability to inhibit IL-6 signaling in appropriate cell-based bioassays, with expected ED₅₀ values typically in the range of 0.05-0.15 μg/mL under standard assay conditions.