Recombinant Human RELMβ

Using Recombinant Human RELMβ in research applications is valuable for studying its roles in immune regulation, inflammation, metabolic processes, epithelial barrier function, and disease mechanisms, particularly in the gastrointestinal tract and metabolic disorders.

Key scientific reasons to use recombinant RELMβ include:

• Dissecting Immune Regulation: RELMβ is a master regulator of type 2 immune responses in the gut, influencing epithelial cell programs, goblet cell function, and the balance between tolerogenic and pathogenic T cell subsets. Recombinant RELMβ enables direct investigation of these pathways in vitro (e.g., organoids, cell lines) and in vivo (animal models).

• Modeling Inflammatory and Metabolic Pathways: RELMβ promotes inflammatory cytokine production (TNFα, IL-1β, IL-6) and activates NF-κB signaling in macrophages, contributing to atherosclerosis and metabolic inflammation. Recombinant protein allows controlled stimulation of immune cells to elucidate these mechanisms.

• Studying Epithelial Barrier and Microbiome Interactions: RELMβ is critical for epithelial barrier integrity and is induced by microbial colonization. Recombinant RELMβ can be used to probe its effects on epithelial cells, tight junctions, and antimicrobial gene expression.

• Cancer and Proliferation Research: RELMβ expression is elevated in proliferative epithelial cells and tumors, suggesting roles in cell proliferation and tumor biology. Recombinant RELMβ is useful for functional assays in cancer cell lines or organoids.

• Biomarker and Diagnostic Development: Recombinant RELMβ serves as a standard or antigen in immunoassays (e.g., ELISA) for quantifying endogenous RELMβ in plasma or tissue samples, supporting biomarker discovery and validation.

• Host Defense Studies: RELMβ acts as a host defense peptide, directly killing bacteria in the gastrointestinal tract, making recombinant protein essential for antimicrobial assays and microbiome research.

• Reproducibility and Experimental Control: Recombinant proteins provide high purity, batch-to-batch consistency, and defined activity, which are critical for reproducible and interpretable experimental results.

In summary, recombinant human RELMβ is a versatile tool for mechanistic studies in immunology, metabolism, epithelial biology, cancer, and host-microbe interactions, enabling precise dissection of its biological functions and therapeutic potential.

Yes, recombinant human RELMβ is suitable for use as a standard for quantification and calibration in ELISA assays. This is the standard approach used in RELMβ detection protocols.

Characteristics of Recombinant RELMβ as an ELISA Standard

Recombinant human RELMβ is produced in E. coli and is supplied as a sterile-filtered, lyophilized powder. The protein is a non-glycosylated, disulfide-linked homodimer with each monomer containing 89 amino acids and a total molecular weight of approximately 19 kDa. The endotoxin level is typically <0.01 ng/μg or <0.1 EU/μg, which is important for maintaining assay reliability.

Standard Curve Preparation

When preparing your standard curve, the recombinant RELMβ should be reconstituted according to manufacturer instructions, as these may be lot-specific. Typical standard curve ranges for RELMβ ELISA assays span from approximately 62.5 to 4000 pg/mL, though the exact range is lot-dependent. The sensitivity of these assays is generally around 22.5 pg/mL.

Validation Considerations

For accurate quantification, ensure that your standard curve demonstrates adequate linearity and reproducibility. The recombinant protein used for calibration should ideally be the same material used in antibody preparation to minimize differences in binding affinity between the standard and endogenous analyte. Additionally, you should validate parallelism and dilution linearity to confirm that your samples yield accurate measurements when diluted and compared against the standard curve.

Best Practices

Use purified or semi-purified recombinant RELMβ for your standard curve. When available, calibrate your standards to reference materials such as NIBSC or WHO standards to ensure harmonized standardization. Generate a fresh standard curve for each set of samples assayed rather than relying on previously established curves.

Recombinant Human RELMβ has been validated in published research for several key applications, primarily in studies of immune regulation, inflammation, epithelial cell biology, and host-microbiome interactions.

Validated Applications:

• Immune Modulation in Gut Epithelium: Recombinant RELMβ has been used to treat murine and human intestinal organoids, demonstrating its role as a master regulator of type 2 epithelial immune programs. It induces expression of antimicrobial and goblet cell-associated genes, modulates tight junction proteins, and influences tolerance to food antigens in vivo and ex vivo.

• Inflammatory Response Assays: In vitro stimulation of human bronchial epithelial cells (16HBE) with recombinant RELMβ increases the expression of inflammatory cytokines such as IL-8 and IL-1β, validating its use in studies of airway inflammation and chronic inflammatory diseases like COPD.

• Antibacterial Activity and Host Defense: RELMβ has been tested for its direct antibacterial properties, showing the ability to kill bacteria by binding to bacterial membranes in vitro, and correlating with in vivo immunity in mouse models.

• Macrophage Activation and Cytokine Induction: Recombinant RELMβ has been used to stimulate macrophages and cell lines (e.g., J774.1), resulting in enhanced expression of inflammatory cytokines (TNFα, IL-1β, IL-6) and activation of the NF-κB pathway, particularly in the context of atherosclerosis and lipid accumulation.

• Biomarker Validation: RELMβ has been measured in human plasma samples to investigate its potential as a biomarker for colorectal cancer and metabolic diseases, supporting its use in ELISA and other immunoassays.

Additional Context:

• RELMβ is secreted by goblet and epithelial cells, and its recombinant form is used to dissect its role in epithelial-microbiome interactions, food allergy models, and airway remodeling.
• It is also implicated in hepatic insulin resistance and metabolic regulation, though direct validation in recombinant protein studies is less frequently reported.

Summary Table of Validated Applications

These applications are supported by direct experimental use of recombinant human RELMβ in published research, confirming its utility in immunological, inflammatory, and biomarker studies.

To reconstitute and prepare Recombinant Human RELMβ protein for cell culture experiments, follow these best-practice steps:

• Centrifuge the vial briefly (3000–3500 rpm for 5 minutes) before opening to collect all lyophilized powder at the bottom.
• Reconstitute the protein by adding sterile water to achieve a concentration of 0.1 mg/mL (100 µg/mL) unless your protocol requires a different concentration. Gently pipette the solution up and down and wash down the sides of the vial to ensure complete recovery. Do not vortex or shake vigorously, as this can denature the protein.
• Allow the protein to dissolve at room temperature for 15–30 minutes with gentle agitation (e.g., slow inversion or gentle pipetting).
• Dilute further as needed for your experiment using cell culture medium or PBS. For best stability, include a carrier protein such as 0.1% BSA, HSA, or FBS in your dilution buffer, especially for long-term storage or low-concentration working solutions.
• Aliquot the reconstituted protein into single-use volumes (≥20 µL) to avoid repeated freeze-thaw cycles, which can reduce activity.
• Storage:
  • Short-term: Store at 4°C for up to one week.
  • Long-term: Store aliquots at –20°C or below. Avoid repeated freeze-thaw cycles.

Summary of critical points:

• Use sterile water for initial reconstitution to 0.1 mg/mL.
• Gently mix; do not vortex.
• Add carrier protein for stability if storing or diluting further.
• Aliquot and freeze for long-term storage.

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