Recombinant Rat Growth Hormone Receptor

Using Recombinant Rat Growth Hormone Receptor (GHR) in research applications provides a highly controlled and specific tool for studying growth hormone signaling, receptor-ligand interactions, and downstream biological effects in rat models. Recombinant GHR enables precise mechanistic studies that are not possible with endogenous or non-recombinant preparations.

Key scientific advantages include:

• Defined molecular composition: Recombinant GHR corresponds to the extracellular domain of the transmembrane receptor, allowing for consistent and reproducible experiments.
• Functional studies: It can be used to investigate receptor dimerization, activation of the JAK/STAT signaling cascade, and modulation of growth hormone (GH) signaling pathways.
• Competitive binding assays: Recombinant GHR acts as a soluble GH-binding protein (GHBP), which can compete with the native receptor for GH, enabling studies of GH signaling inhibition or enhancement.
• Neutralization and inhibition assays: It is useful for evaluating the effects of antibodies or small molecules that target GHR, as well as for dissecting the role of GHR in cell proliferation and other GH-mediated processes.
• Positive control and immunogen: Recombinant GHR serves as a reliable positive control in Western blot, ELISA, and other immunoassays, and can be used as an immunogen for antibody production.
• Protein-protein interaction studies: It facilitates the analysis of GH-GHR binding kinetics, receptor cross-talk, and downstream signaling events in vitro.

Typical applications include:

• Cell signaling research: Dissecting the molecular mechanisms of GH action, including receptor activation and downstream effects.
• Pharmacological studies: Screening and characterization of GHR agonists, antagonists, and neutralizing antibodies.
• Disease modeling: Investigating the role of GHR in metabolic, growth, and endocrine disorders in rat models.
• Biomarker development: Using recombinant GHR as a standard or control in assays quantifying GHR or GHBP levels.

In summary, recombinant rat GHR provides a standardized, versatile reagent for mechanistic, pharmacological, and translational research focused on growth hormone biology in rat systems.

A recombinant rat growth hormone receptor (GHR) can be used as a standard for quantification or calibration in ELISA assays, but only under specific conditions: the ELISA must be validated to recognize the recombinant form with comparable affinity to the native protein, and the standard curve must be constructed using the same recombinant protein preparation.

Key considerations:

• ELISA specificity: Many commercial ELISA kits for rat GHR are designed to detect the native protein in biological samples. Some kits explicitly state that their standards are based on recombinant GHR, and these kits provide a standard curve using the recombinant protein. If your ELISA kit uses recombinant GHR as its calibrator, you can use your recombinant protein preparation for quantification, provided it matches the kit’s standard in purity and structure.

• Validation required: If your recombinant GHR differs in glycosylation, folding, or sequence from the native protein or the kit’s standard, you must validate that your ELISA detects it with the same sensitivity and specificity. Differences in post-translational modifications or expression systems can affect antibody recognition and quantification accuracy.

• Standard curve construction: To use recombinant GHR as a standard, prepare a dilution series covering the assay’s dynamic range (e.g., 0.14–100 ng/mL for some kits). Run these standards in parallel with your samples to generate a calibration curve (OD vs. concentration), then interpolate sample concentrations from this curve.

• Documentation: Always check the ELISA kit’s instructions and technical datasheet. Some kits caution against using recombinant proteins from other sources as standards due to potential differences in antibody recognition. If the kit’s standard is recombinant GHR, and your preparation is equivalent, it is suitable for calibration.

Best practices:

• Confirm the recombinant GHR’s identity, purity, and concentration using independent methods (e.g., SDS-PAGE, BCA assay).
• Validate parallelism between the standard curve generated with your recombinant GHR and the kit’s standard curve using native protein, if possible.
• Document all validation steps and controls for reproducibility.

Summary Table: Recombinant GHR as ELISA Standard

In conclusion, recombinant rat GHR can be used as a standard for ELISA quantification if the assay is validated for this purpose and the recombinant protein matches the kit’s standard. Always consult the kit documentation and perform necessary validation experiments.

Recombinant rat growth hormone receptor (GHR) has been validated in published research primarily for studies of growth hormone signaling, receptor-ligand interactions, and downstream biological effects in rat models. The applications focus on mechanistic, pharmacological, and physiological investigations.

Key validated applications include:

• Ligand binding and receptor activation studies: Recombinant rat GHR is used to study the binding of growth hormone (GH) and the resulting receptor dimerization and activation of downstream signaling pathways, such as the JAK/STAT cascade. These studies are foundational for understanding GH signaling mechanisms in rats.

• Pharmacological and toxicological research: Recombinant rat GH and its receptor have been used to evaluate the biological activity, safety, and potential carcinogenicity of GH in rats. These studies measure endpoints such as body weight gain, serum IGF-1 levels, and long-term safety (e.g., tumor incidence) in hypophysectomized or normal rats.

• Gene expression and receptor regulation: Research has validated the use of recombinant GHR to assess changes in GHR mRNA and protein expression in various physiological and pathological states, such as liver cirrhosis, where GHR expression is downregulated and can be modulated by GH treatment.

• Signal transduction and functional assays: Recombinant GHR is used in cell-based assays to investigate the activation of intracellular signaling pathways (e.g., JAK/STAT) following GH binding, as well as to study the effects of GH or GHR agonists/antagonists on cell proliferation, apoptosis, and metabolic functions.

• Structural and mechanistic studies: Recombinant GHR is employed in structural biology to elucidate the molecular basis of receptor activation and ligand specificity, which informs drug design and therapeutic targeting.

• In vivo physiological studies: Recombinant GHR and GH are used in animal models to study growth, metabolism, and tissue-specific effects, including the modulation of reproductive development, organ growth, and metabolic parameters.

Summary Table: Validated Applications of Recombinant Rat GHR

Note: While some studies use recombinant human GH or GHR in rat models, the applications above specifically reference recombinant rat GHR or its direct use in rat systems. For any specialized application (e.g., therapeutic development, disease modeling), validation should be confirmed for the specific experimental context.

To reconstitute and prepare Recombinant Rat Growth Hormone Receptor (GHR) protein for cell culture experiments, follow these general best practices based on manufacturer protocols and scientific recommendations:

1. Reconstitution

• Reconstitution Buffer:
  Most recombinant rat GHR proteins (especially His-tagged, lyophilized forms) should be reconstituted in sterile PBS (pH 7.4) or sterile deionized water.

  • Example:
    • Reconstitute in 10 mM PBS (pH 7.4) to a concentration of 0.1–1.0 mg/mL (Cloud-Clone, ACROBiosystems).
    • Some protocols recommend sterile deionized water to a final concentration of 200 µg/mL (Abcam, similar for rat proteins).
    • Avoid using buffers with strong denaturants or detergents unless specified.

• Procedure:

  • Gently add the buffer to the lyophilized protein vial.
  • Let the vial sit at room temperature for 30–60 minutes to allow the protein to dissolve.
  • Do not vortex—gently mix by pipetting up and down or by slow rotation.
  • Avoid vigorous shaking to prevent protein denaturation.

2. Storage and Handling

• Short-term:
  • Reconstituted protein can be stored at 4–8°C for 2–7 days.
• Long-term:
  • Aliquot the reconstituted protein and store at ≤ –20°C (preferably ≤ –80°C) for up to 3 months.
  • Avoid repeated freeze-thaw cycles, as this can reduce activity and stability.

3. Preparation for Cell Culture

• Dilution:

  • Dilute the reconstituted protein in cell culture medium (e.g., DMEM, RPMI) or serum-free medium just before use.
  • Use low-binding tubes and pipette tips to minimize protein loss due to adsorption.

• Concentration:

  • Typical working concentrations for bioactivity assays (e.g., inhibition of cell proliferation) range from 0.5–2 µg/mL in the presence of growth hormone (based on ED50 values).
  • Always refer to the specific product’s datasheet for recommended concentrations and assay conditions.

• Controls:

  • Include appropriate controls (e.g., vehicle-only, untreated cells) to ensure experimental validity.

4. Additional Tips

• Sterility:
  • Use sterile techniques throughout to avoid contamination, especially for long-term cell culture experiments.
• Protectants:
  • Some lyophilized proteins include trehalose, mannitol, or Tween 80 as protectants. These are generally safe for cell culture but may need to be considered in sensitive assays.
• Activity Assay:
  • If possible, validate the protein’s activity using a relevant cell-based assay (e.g., inhibition of growth hormone-induced proliferation).

Summary Protocol

1. Reconstitute lyophilized rat GHR protein in sterile PBS (pH 7.4) to 0.1–1.0 mg/mL.
2. Gently mix and let sit at room temperature for 30–60 minutes.
3. Aliquot and store at ≤ –20°C for long-term use.
4. Dilute in cell culture medium to desired concentration (e.g., 0.5–2 µg/mL) before adding to cells.
5. Use sterile, low-binding materials and avoid repeated freeze-thaw cycles.

Always consult the product-specific datasheet or certificate of analysis (CoA) for exact reconstitution and storage instructions, as protocols may vary slightly between manufacturers.