Recombinant Human GDF-15 – D-variant

Using Recombinant Human GDF-15 D-variant in research is essential when you need to study the biological effects, signaling, or measurement of the naturally occurring D-variant (H202D, Aspartate at position 202) of GDF-15, which is a common human polymorphism. The D-variant is functionally equivalent to the wild-type in terms of bioactivity, but it can significantly affect immunoassay measurements and is relevant for studies on disease association, biomarker development, and therapeutic targeting.

Key reasons to use the D-variant in your research:

• Genetic Relevance: The D-variant (H202D) is a naturally occurring polymorphism in the human population, with clinical studies showing it can influence disease risk and prognosis, particularly in cancer and cardiometabolic diseases.
• Assay Accuracy: The D-variant can cause underestimation of GDF-15 levels in certain immunoassays (notably those using R&D Systems reagents), which is critical for biomarker studies and clinical research. Using the D-variant protein allows you to calibrate assays or validate findings in populations carrying this allele.
• Functional Studies: The D-variant has been shown to have indistinguishable bioactivity compared to the wild-type (H-variant), making it suitable for mechanistic studies on GDF-15’s roles in inflammation, metabolism, immune regulation, and tissue injury.
• Disease Association: The D-variant is associated with altered risk and prognosis in several diseases, including colorectal and prostate cancer, making it important for genotype-phenotype correlation studies.
• Therapeutic Research: GDF-15 (including the D-variant) is a target for therapies in obesity, metabolic syndrome, and cancer, and recombinant protein is used in preclinical models to study its effects on weight loss, glucose metabolism, and immune modulation.

Best practices:

• Use the D-variant when studying populations or diseases where the H202D polymorphism is prevalent or relevant.
• Employ the D-variant for assay calibration to ensure accurate quantification in genetic subgroups.
• Confirm the biological activity of your recombinant preparation, as most commercial sources report activity in cell-based assays (e.g., inhibition of DU-145 cells).

Summary:
Use Recombinant Human GDF-15 D-variant for research requiring genetic specificity, assay accuracy, or functional studies related to the H202D polymorphism, especially in biomarker, metabolic, and immunological research contexts.

Recombinant Human GDF-15 D-variant can be used as a standard for quantification or calibration in ELISA assays, provided the assay is validated to detect this specific variant. The suitability depends on the ELISA's antibody specificity and calibration protocol.

Key considerations:

• Variant specificity: The D-variant of GDF-15 contains a His→Asp substitution at position 7. Some ELISA kits are designed to detect total GDF-15 (including both H and D variants), while others are H-specific and do not recognize the D-variant. For accurate quantification, ensure your ELISA detects the D-variant. Total GDF-15 ELISAs have been shown to quantify both recombinant and natural GDF-15 variants equally.

• Calibration standards: Many commercial ELISA kits use recombinant human GDF-15 (often the D-variant) as a calibration standard. Recovery experiments demonstrate that recombinant GDF-15 can be spiked into biological samples and accurately measured by validated ELISA protocols.

• Validation: Before using the D-variant as a standard, confirm that your assay's antibodies recognize the D-variant with similar affinity as endogenous GDF-15 in your sample matrix. If using a kit, consult the manufacturer's documentation to verify compatibility.

• Documentation: Published protocols and kit inserts indicate that recombinant GDF-15 (including D-variant) is routinely used for calibration and recovery studies in ELISA assays for total GDF-15.

Best practices:

• Validate the standard curve using the recombinant D-variant in your specific ELISA setup.
• Include recovery and dilution linearity controls to confirm assay performance with the D-variant.
• If your assay is H-specific, the D-variant will not be detected and is unsuitable as a standard.

Summary Table:

In conclusion: You can use recombinant human GDF-15 D-variant as a standard for quantification or calibration in ELISA assays if your assay is validated for total GDF-15 detection and not restricted to the H-variant. Always verify antibody specificity and assay documentation before proceeding.

Recombinant Human GDF-15 - D-variant has been validated for several applications in published research, primarily related to its biological activity and functional roles in disease models. Key applications include:

1. Protection of Pancreatic β Cells:
   Recombinant GDF-15 (including the D-variant) has been shown to protect human pancreatic β cells against pro-inflammatory cytokine-mediated apoptosis and cellular stress. Pretreatment with rGDF-15 reduces insulitis and increases β cell survival, suggesting its potential as a therapeutic agent for type 1 diabetes (T1D) prevention and management .

2. Induction of Muscle Atrophy:
   GDF-15 protein, including recombinant forms, can directly induce muscle atrophy in cultured C2C12 myotubes by regulating apoptotic pathways such as Bcl-2/caspase-3. This application is relevant for studying cancer cachexia and muscle wasting disorders .

3. Anti-inflammatory and Immune-Regulatory Effects:
   GDF-15 has demonstrated anti-inflammatory functions in various models, including sepsis, where GDF-15 knockout mice exhibit heightened inflammatory responses. Recombinant GDF-15 is used to study immune checkpoint regulation and its potential as a target for cancer immunotherapy .

4. Metabolic Regulation and Weight Loss:
   Treatment with recombinant GDF-15 in rodent models fed a high-fat diet results in weight loss, reduced liver steatosis, and improved glycemic control. These studies highlight its role in appetite regulation, energy expenditure, and metabolic health .

5. Biomarker Studies:
   The D-variant of GDF-15 is used in biomarker research, particularly for early detection of heart disease and heart failure. It is also studied as a prognostic marker for diabetes and cardiovascular disease .

6. Measurement and Assay Validation:
   The D-variant has been specifically studied for its impact on immunoassay measurements, as it can affect the accuracy of GDF-15 quantification in clinical and research settings. This is important for interpreting results in studies involving GDF-15 as a biomarker [8, 11].

These applications demonstrate the versatility of Recombinant Human GDF-15 - D-variant in both basic research and translational studies, particularly in the fields of diabetes, cancer, metabolism, and cardiovascular disease.

To reconstitute and prepare Recombinant Human GDF-15 D-variant protein for cell culture experiments, follow these steps:

• Centrifuge the vial briefly before opening to ensure all lyophilized material is at the bottom.
• Reconstitute the protein by adding sterile water to achieve a final concentration of 0.1 mg/mL. Gently pipette the solution down the sides of the vial to dissolve the protein completely. Do not vortex, as vigorous mixing can denature the protein.
• Allow several minutes for complete dissolution. If necessary, gently swirl or invert the vial.
• For long-term storage, aliquot the reconstituted protein into working volumes, dilute with a carrier protein such as 0.1% BSA in PBS, and store at -80°C. Avoid repeated freeze-thaw cycles to preserve protein integrity.

Preparation for cell culture experiments:

• Before use, dilute the reconstituted stock solution to the desired working concentration using sterile cell culture medium or buffer compatible with your assay.
• If your application is sensitive to endotoxin, confirm the endotoxin level is below 0.1 EU/µg, which is typical for recombinant GDF-15 D-variant preparations.
• For bioactivity assays, typical working concentrations range from ng/mL to µg/mL, depending on cell type and experimental design.

Additional notes:

• If your protocol or supplier recommends a different reconstitution buffer (e.g., 4 mM HCl, 5 mM acetic acid, or PBS), use that buffer instead of water, as some recombinant proteins may require acidic conditions for optimal solubility and stability.
• Always consult the specific product datasheet for any unique instructions regarding buffer composition or handling.

Summary of best practices:

• Centrifuge vial, reconstitute in sterile water (or specified buffer) at 0.1 mg/mL, gentle mixing, aliquot with carrier protein for storage, avoid freeze-thaw cycles, and dilute to working concentration in cell culture medium for experiments.