Recombinant Human Relaxin-3

Recombinant Human Relaxin-3 is used in research applications to study its unique roles as a neuropeptide in the central nervous system, particularly its involvement in neuroendocrine regulation, stress response, feeding behavior, and potential therapeutic targeting of its receptor RXFP3.

Key reasons to use recombinant human Relaxin-3 in research include:

• Selective Agonist for RXFP3: Relaxin-3 is the only known high-affinity endogenous ligand for the G-protein-coupled receptor RXFP3 (also known as GPCR135), making it essential for dissecting RXFP3-mediated signaling pathways.
• Neuroendocrine and Behavioral Studies: Relaxin-3/RXFP3 signaling modulates hypothalamic function, influencing arousal, stress, feeding, metabolism, and anxiety-related behaviors. Recombinant Relaxin-3 enables controlled studies of these effects in vitro and in vivo.
• Mechanistic Insights: Recombinant protein allows for precise structure-function analyses, such as mapping critical residues for receptor binding and activation, and understanding conformational dynamics upon receptor interaction.
• Therapeutic Target Validation: RXFP3 is a potential target for treating neuroendocrine and behavioral disorders (e.g., anxiety, obesity). Recombinant Relaxin-3 is used to validate pharmacological effects and downstream signaling relevant to drug development.
• Reproducibility and Consistency: Recombinant production ensures batch-to-batch consistency, purity, and defined activity, which are critical for reproducible experimental outcomes and mechanistic studies.

Additional relevant information:

• Functional Assays: Recombinant Relaxin-3 is used to activate RXFP3 in cell-based assays, modulate cAMP signaling, and study downstream gene expression changes.
• In Vivo Applications: Central administration of recombinant Relaxin-3 in animal models has demonstrated effects on food intake, body weight, stress response, and anxiety-like behaviors, supporting its physiological relevance.
• Cross-Reactivity: While Relaxin-3 can also interact with RXFP1 (LGR7) and RXFP4, its highest affinity and most specific effects are mediated via RXFP3, distinguishing it from other relaxin family peptides.

In summary, recombinant human Relaxin-3 is a critical tool for elucidating the physiological and pathophysiological roles of the relaxin-3/RXFP3 system, enabling both basic research and translational studies targeting neuroendocrine and behavioral disorders.

Recombinant Human Relaxin-3 can be used as a standard for quantification or calibration in ELISA assays, provided it is properly validated and matched to your assay system.

Key considerations and supporting details:

• Intended Use: Recombinant Human Relaxin-3 is commonly used as an ELISA standard, especially when formulated with BSA for stability. Many ELISA kits and protocols specify recombinant proteins as standards for generating calibration curves.

• Validation: It is essential to validate the recombinant standard in your specific ELISA system. This includes confirming that the recombinant protein is immunologically equivalent to the endogenous analyte and that it produces a reliable, linear standard curve within the assay's dynamic range. Differences in protein folding, post-translational modifications, or sequence variants can affect antibody recognition and quantification accuracy.

• Calibration: Standards provided in commercial ELISA kits are typically calibrated against a master calibrator. If you use a recombinant protein not supplied with your kit, you should assign its value based on measurement in your ELISA rather than relying solely on the mass stated on the vial. This helps account for any discrepancies in immunoreactivity or protein quantification.

• Formulation: For ELISA, the recombinant protein with BSA is recommended to enhance stability and reproducibility. Carrier-free formulations are available but may be less stable and require careful handling.

• Dilution and Handling: Large dilution steps (from μg/mL to pg/mL) are often required to place the recombinant standard on the ELISA curve, which can introduce error. Accurate pipetting and thorough mixing are critical.

• Documentation: Always document the source, lot number, and concentration assignment method for your standard to ensure reproducibility and traceability.

Best Practices:

• Validate the recombinant Relaxin-3 standard in your assay by running parallel curves with known controls.
• Assign concentrations based on ELISA measurements, not just vial label mass.
• Use the same buffer and diluent conditions for standards and samples to minimize matrix effects.

Summary Table: Recombinant Relaxin-3 as ELISA Standard

If you follow these guidelines, recombinant Human Relaxin-3 is suitable as a standard for ELISA quantification and calibration.

Recombinant Human Relaxin-3 has been validated in published research for several key applications, primarily in neurobiology and receptor pharmacology. The most common validated uses include:

• Receptor binding and activation assays: Relaxin-3 is widely used to study its interaction with its primary receptor, RXFP3, and to a lesser extent RXFP4 and RXFP1, using cell-based assays to measure downstream signaling events such as cAMP modulation. These assays often employ recombinant relaxin-3 to characterize receptor pharmacology, ligand specificity, and mutagenesis effects on receptor activation.

• In vivo functional studies: Recombinant relaxin-3 has been administered centrally (e.g., intracerebroventricular injection) in rodent models to investigate its physiological roles in the brain. Validated applications include:

  • Feeding behavior and metabolism: Chronic or acute administration increases food intake and body weight, implicating relaxin-3 in energy homeostasis.
  • Stress and anxiety modulation: Relaxin-3 administration has been shown to reduce anxiety-like behaviors in established behavioral paradigms (elevated plus maze, light-dark box, shock probe-burying test).
  • Neuroendocrine regulation: Studies have used recombinant relaxin-3 to probe its effects on hypothalamic-pituitary axis hormones, such as decreasing plasma thyroid-stimulating hormone (TSH) levels.

• Structure-function and mutagenesis studies: Recombinant relaxin-3 is used in mutagenesis experiments to dissect the molecular determinants of receptor binding and activation, including alanine scanning and site-directed mutagenesis of both ligand and receptor.

• Pharmacological tool development: Relaxin-3 and its analogs serve as reference ligands for the development and validation of RXFP3-targeted therapeutics, including small molecules and peptide analogs for potential treatment of CNS disorders (anxiety, depression, obesity, binge eating, alcohol use disorder).

• ELISA and biochemical assays: Recombinant relaxin-3 is used as a standard or control in immunoassays to quantify endogenous or exogenous relaxin-3 levels in biological samples.

Summary Table: Validated Applications of Recombinant Human Relaxin-3

Additional context:

• Most studies use recombinant relaxin-3 in in vitro cell-based assays or in vivo rodent models, focusing on CNS-related endpoints.
• There is strong evidence for its role in modulating feeding, stress, arousal, and neuroendocrine function, making it a valuable tool for both basic and translational neuroscience research.
• Recombinant relaxin-3 is not typically used in clinical diagnostics or as a therapeutic agent itself, but rather as a research reagent.

If you require protocols or more detailed information on a specific application, please specify the context.

To reconstitute and prepare Recombinant Human Relaxin-3 for cell culture experiments, dissolve the lyophilized protein at 100 μg/mL in sterile PBS containing at least 0.1% human or bovine serum albumin (BSA) as a carrier protein. This helps prevent protein loss due to adsorption and stabilizes the protein during handling.

Step-by-step protocol:

• Centrifuge the vial briefly before opening to ensure all powder is at the bottom.
• Add sterile PBS with 0.1% BSA to achieve a final concentration of 100 μg/mL.
• Gently mix by swirling or inverting; avoid vigorous shaking or vortexing to prevent foaming and protein denaturation.
• Allow the solution to sit at room temperature for 15–30 minutes with gentle agitation to ensure complete dissolution.
• If particulates remain, continue gentle mixing at room temperature or at 4°C for up to several hours.

Aliquoting and storage:

• Aliquot the reconstituted protein to avoid repeated freeze-thaw cycles, which can degrade the protein.
• For short-term storage (up to 1 week), keep at 2–8°C.
• For long-term storage (up to 3 months), store aliquots at –20°C to –70°C.
• Always use a manual defrost freezer for long-term storage and avoid repeated freeze-thaw cycles.

Dilution for cell culture:

• For working concentrations, further dilute the stock solution in cell culture medium containing carrier protein (e.g., 0.1% BSA) to minimize adsorption and maintain activity.
• If using serum-free media, consider using a non-animal carrier such as trehalose to avoid introducing animal proteins.

Additional notes:

• Always consult the specific product datasheet or Certificate of Analysis for any unique instructions, as formulations may vary.
• If the protein is to be used in sensitive applications (e.g., in vivo or serum-free culture), ensure the carrier protein is compatible with your system.

This protocol ensures optimal solubility, stability, and biological activity of recombinant human Relaxin-3 for cell culture experiments.