Recombinant Human Prolactin

Recombinant Human Prolactin (r-hPRL) is used in research applications primarily because it provides a biologically active, consistent, and well-defined form of prolactin that enables controlled studies of prolactin’s physiological and pathological roles, particularly in lactation, immunology, and cell signaling.

Key reasons to use recombinant human prolactin in research include:

• Lactation Studies: r-hPRL is essential for investigating the mechanisms of lactation, as it can restore or augment milk production in models of prolactin deficiency or lactation insufficiency. It has been shown to increase milk volume and induce changes in milk composition that mirror normal lactogenesis, making it valuable for studying maternal and neonatal nutrition, especially in preterm or prolactin-deficient contexts.

• Standardization in Assays: Recombinant prolactin serves as a reliable standard in immunodiagnostic assays, reducing variability and eliminating interference from heterogeneous serum isoforms. This improves the accuracy, sensitivity, and specificity of prolactin measurements in clinical and research settings.

• Cellular and Molecular Research: r-hPRL is used to study prolactin receptor signaling, cell proliferation, survival, and migration, including its roles in cancer biology and immune modulation. It enables experiments that require precise control over hormone concentration and isoform, which is not possible with native pituitary extracts.

• Immunology and Regeneration: Research has shown that recombinant prolactin can promote immune and myeloid reconstitution after bone marrow transplantation in animal models, supporting studies in hematopoiesis and immune system recovery.

• Ethical and Practical Advantages: Recombinant production eliminates the need for human pituitary tissue, which is scarce and raises ethical concerns. Recombinant proteins are available in large, consistent lots, ensuring reproducibility and scalability for research and assay development.

• Therapeutic Research: r-hPRL is being investigated as a potential therapeutic for lactation insufficiency and prolactin deficiency, providing a translational bridge from basic research to clinical application.

In summary, recombinant human prolactin is preferred in research for its biological activity, consistency, assay standardization, and ethical production, supporting a wide range of studies from basic science to clinical translation.

Recombinant human prolactin can be used as a standard for quantification or calibration in ELISA assays, provided it is appropriately validated and matched to the assay system. The suitability depends on the specific ELISA kit and the intended application.

Key considerations and supporting details:

• WHO Reference Standard: The World Health Organization (WHO) has established both native and recombinant human prolactin preparations as international standards for immunoassay calibration. The recombinant standard (coded 97/714) is specifically intended as a reference reagent for immunoassays, with a defined content and validated performance. This demonstrates that recombinant prolactin can serve as a calibration standard if it is traceable to an international reference and its commutability with clinical samples is established.

• Assay Compatibility: Many commercial ELISA kits for human prolactin quantification use recombinant prolactin as their standard, and these kits are validated to ensure that recombinant and native prolactin produce parallel standard curves. This indicates that, in principle, recombinant prolactin is suitable for use as a standard in quantitative ELISA, provided the assay is validated for this purpose.

• Validation Required: If you are using a recombinant prolactin preparation not specifically provided or validated by your ELISA kit manufacturer, you must ensure:

  • The recombinant protein is of high purity and correctly folded.
  • Its concentration is accurately determined.
  • The standard curve generated with the recombinant protein is parallel to that obtained with native prolactin or the kit’s recommended standard.
  • The recombinant standard is traceable to an international reference, if clinical comparability is required.

• Potential Limitations: Some recombinant proteins may differ in glycosylation or folding compared to native serum prolactin, which can affect antibody recognition in certain assays. Therefore, it is essential to confirm that your ELISA detects recombinant and native prolactin equivalently.

• Best Practices:

  • Use recombinant prolactin preparations that are traceable to the WHO International Standard for clinical or regulatory work.
  • Validate the standard curve for linearity, parallelism, and recovery using both recombinant and native samples, if possible.
  • Consult the ELISA kit documentation to confirm compatibility with recombinant standards.

Summary:
You can use recombinant human prolactin as a standard for ELISA quantification if it is validated for your assay, ideally traceable to an international standard, and produces parallel standard curves with native prolactin. Always verify assay compatibility and perform appropriate validation steps to ensure accurate quantification.

Recombinant Human Prolactin has been validated for a range of applications in published research, primarily in the following areas:

• Bioassays: Used to assess prolactin’s biological activity, including its effects on cell proliferation, signaling pathways (such as PI3K/Akt, MAPK/ERK, and NF-κB), and hormone receptor activation in various cell types, including breast cancer cells, immune cells, and organoids.
• Cell Culture: Applied to study prolactin’s effects on cell differentiation, proliferation, and gene expression in cultured cells, such as corneal stromal fibroblasts, immune cells, and mammary epithelial cells.
• Lactation Studies: Used in clinical and preclinical studies to treat lactation insufficiency and prolactin deficiency in mothers, demonstrating increased milk volume and changes in breast milk composition consistent with normal lactation.
• Immunological Research: Investigated for its immunomodulatory effects on T cells, B cells, macrophages, and its role in autoimmune disease models.
• Cancer Research: Utilized to study prolactin’s role in breast and prostate cancer development, tumor growth, and signaling pathway activation in cancer cell lines.
• Cell Protection and Transplantation: Explored for protective effects on pancreatic β-cells, neurons, and during islet cell and liver transplantation.
• Adipocyte Differentiation: Used to evaluate effects on gene expression related to adipocyte differentiation and metabolism in cell models (primarily with mouse prolactin, but methods are analogous).

Key validated applications in published research:

• Functional bioassays (e.g., cell proliferation, receptor activation)
• Cell culture supplementation for mechanistic studies
• Clinical trials for lactation induction and breast milk composition improvement
• Immunological assays (e.g., cytokine production, immune cell modulation)
• Cancer cell signaling and proliferation studies
• Cell protection assays in transplantation and oxidative stress models

These applications are supported by peer-reviewed studies and clinical trials, confirming the protein’s activity and utility in both basic and translational research.

To reconstitute and prepare Recombinant Human Prolactin for cell culture experiments, dissolve the lyophilized protein in sterile distilled water or an appropriate buffer to a concentration of 0.1 mg/mL, then dilute to your desired working concentration in cell culture medium.

Step-by-step protocol:

• Centrifuge the vial briefly before opening to ensure all powder is at the bottom.
• Add sterile distilled water (or buffer, e.g., 10 mM sodium phosphate, 150 mM NaCl, pH 7.4) to achieve a stock concentration of 0.1 mg/mL.
• Gently mix by pipetting up and down or swirling; do not vortex, as this may denature the protein.
• Allow the protein to fully dissolve for 15–30 minutes at room temperature with gentle agitation.
• If required for stability, add a carrier protein such as 0.1% BSA or HSA, especially for long-term storage or low working concentrations.
• Aliquot the reconstituted stock to avoid repeated freeze-thaw cycles, and store at -80°C for long-term use.
• Dilute the stock solution in cell culture medium or buffer immediately before use to the final concentration needed for your experiment.

Additional notes:

• Avoid vigorous mixing or foaming, which can cause protein denaturation.
• Use polypropylene tubes for storage to minimize protein adsorption.
• For cell culture, ensure the final buffer is compatible with your cells (e.g., PBS or culture medium).
• Always check the product datasheet for specific recommendations regarding buffer composition and reconstitution volume, as formulations may vary between preparations.

Example preparation for cell culture:

1. Centrifuge the vial briefly.2. Add 100 µL sterile distilled water to the vial for a 0.1 mg/mL stock solution.3. Gently mix and allow to dissolve for 15–30 minutes.4. Aliquot and store at -80°C.5. Dilute the stock in cell culture medium to the desired working concentration (e.g., 1–100 ng/mL).

This protocol ensures optimal solubility and biological activity of recombinant human prolactin for cell-based assays.