Recombinant Human Midkine

Recombinant Human Midkine (rhMK) is a multifunctional growth factor widely used in research due to its roles in cell proliferation, survival, migration, angiogenesis, tissue repair, and disease modeling. Its use is particularly valuable for studies in developmental biology, regenerative medicine, cancer research, and neurobiology.

Key reasons to use recombinant human midkine in research applications:

• Potent Mitogenic and Survival Effects: rhMK stimulates proliferation of various cell types, including articular chondrocytes, endothelial cells, fibroblasts, and neuronal cells, both in vitro and in vivo. For example, rhMK significantly increases chondrocyte proliferation and cartilage thickness, making it useful for cartilage repair and regenerative studies.

• Neurotrophic and Neuroprotective Properties: Midkine is highly expressed during embryogenesis and after CNS injury, where it promotes neuronal survival, repair, and regeneration. It is a key mediator in neuro-immune crosstalk and is being explored as a therapeutic target for neurodegenerative diseases and acute CNS injuries.

• Angiogenesis and Tissue Repair: rhMK promotes angiogenesis and tissue repair, making it relevant for wound healing, ischemia models, and studies of vascular biology.

• Cancer Research: Midkine is overexpressed in many cancers and contributes to tumor growth, metastasis, angiogenesis, and therapeutic resistance. It serves as a biomarker for cancer progression and a potential therapeutic target, making rhMK essential for cancer biology and drug development studies.

• Immunomodulation: Midkine modulates immune responses, particularly in the context of inflammation, autoimmunity, and tumor microenvironments. It is involved in the recruitment and activation of immune cells, making it relevant for immunology research.

• Experimental Control and Reproducibility: Using recombinant human midkine ensures a defined, consistent, and biologically active protein, which is critical for reproducible results in cell-based assays, animal models, and biochemical studies.

• Versatility in Applications: rhMK is used in a variety of assays, including cell proliferation, migration, survival, angiogenesis, and gene expression studies. It is also used as a standard in ELISA and other immunoassays.

In summary, recombinant human midkine is a powerful tool for dissecting mechanisms of cell growth, tissue repair, neuroprotection, angiogenesis, cancer progression, and immune regulation, and is essential for translational research in these fields.

Recombinant Human Midkine can be used as a standard for quantification or calibration in ELISA assays, provided it is of high purity and its concentration is accurately determined. This is a common practice in quantitative ELISA protocols, where recombinant proteins serve as standards to generate calibration curves for analyte quantification.

Essential context and best practices:

• Purity and Quantification: The recombinant Midkine should be highly purified, and its concentration must be precisely measured, ideally by an orthogonal method such as HPLC or UV absorbance. Impurities or inaccurate concentration can compromise the reliability of your standard curve.

• Standard Curve Preparation: Prepare serial dilutions of the recombinant Midkine to cover the expected range of your assay (typically 0–2000 pg/mL, but this may vary depending on your kit and sample type). Follow the specific protocol provided with your ELISA kit for dilution buffers and curve range.

• Kit Compatibility: Most commercial ELISA kits for Human Midkine are validated to detect both natural and recombinant forms, and their protocols include recombinant Midkine as the standard. However, always confirm that your recombinant protein matches the form and sequence recognized by the kit antibodies.

• Calibration and Validation: If you are using a recombinant standard not supplied with your ELISA kit, it is recommended to calibrate it against a mass-calibrated reference standard if available, and validate its performance in your assay system. This ensures accuracy and reproducibility.

• Carrier Proteins: Some recombinant proteins are supplied with carrier proteins (e.g., BSA) to improve stability. For ELISA standard use, carrier-free preparations are preferred to avoid interference.

• Documentation: Refer to your ELISA kit’s protocol for specific instructions on standard preparation, curve fitting, and quantification procedures.

Additional considerations:

• If your recombinant Midkine is not supplied with your ELISA kit, ensure it is compatible with the kit’s antibodies and detection system.
• Avoid using standards from different sources interchangeably without validation, as differences in protein folding, post-translational modifications, or sequence variants may affect assay performance.
• Always document the lot number, concentration, and preparation method of your recombinant standard for reproducibility.

In summary, recombinant Human Midkine is suitable as a standard for ELISA quantification if it is pure, accurately quantified, and compatible with your assay system. Proper validation and adherence to best practices are essential for reliable results.

Recombinant Human Midkine has been validated for a range of applications in published research, including functional assays, ELISA, Western blot, immunohistochemistry, and cell-based proliferation and migration studies.

Key validated applications and research uses include:

• Functional Assays: Recombinant human midkine (rhMK) has been used to stimulate proliferation of various cell types, such as articular chondrocytes, in both in vitro and in vivo models. These studies often employ assays like BrdU incorporation and MTT to measure cell proliferation, as well as histological evaluation for tissue-level effects.
• ELISA (Enzyme-Linked Immunosorbent Assay): rhMK is commonly used as a standard or analyte in ELISA to quantify midkine levels in biological samples.
• Western Blot: Detection and quantification of midkine protein in cell or tissue lysates using Western blotting has been validated.
• Immunohistochemistry: rhMK is used to validate antibody specificity and to study midkine localization in tissue sections.
• Cell Proliferation and Migration Assays: Published studies have demonstrated that rhMK promotes proliferation and migration in multiple cell types, including chondrocytes, endothelial cells, fibroblasts, and cancer cells, supporting its use in cell biology and cancer research.
• Angiogenesis Assays: rhMK has been shown to promote angiogenesis in vitro and in vivo, making it relevant for studies of vascular biology and tumorigenesis.
• Cancer Biomarker and Immunology Research: rhMK is used in studies investigating its role as a cancer biomarker, its effects on tumor progression, chemoresistance, and its immunomodulatory properties.

Additional research areas where recombinant human midkine has been applied include:

• Neurobiology: Studies on neuroprotection, neuroinflammation, and neurodegenerative diseases.
• Autoimmune and Inflammatory Disease Models: Investigation of midkine’s role in diseases such as rheumatoid arthritis and systemic lupus erythematosus.
• Tissue Regeneration and Repair: Research on cartilage repair and tissue regeneration, leveraging midkine’s proliferative and anti-apoptotic effects.

These applications are supported by both direct experimental validation and extensive use in peer-reviewed research, demonstrating the versatility of recombinant human midkine in diverse biological and biomedical studies.

To reconstitute and prepare Recombinant Human Midkine protein for cell culture experiments, follow these best-practice steps based on current protocols and technical recommendations:

1. Preparation Before Reconstitution

• Centrifuge the vial at 3000 rpm for 5 minutes to ensure all lyophilized powder is at the bottom before opening.
• Work in a sterile environment to avoid contamination.

2. Reconstitution

• Solvent: Use sterile distilled water or sterile PBS (phosphate-buffered saline) for reconstitution.
• Concentration: Reconstitute to a concentration of at least 100 μg/mL (0.1 mg/mL) or up to 1 mg/mL, depending on your experimental requirements.
• Procedure:
  • Add the appropriate volume of solvent directly to the vial.
  • Gently swirl or invert the vial to mix. Do not vortex or shake vigorously, as this can denature the protein and reduce biological activity.
  • Allow the solution to sit at room temperature for 15–30 minutes to ensure complete dissolution.

3. Dilution for Cell Culture

• For working concentrations, further dilute the reconstituted stock in cell culture medium or buffer.
• To minimize protein loss due to adsorption, dilute using a buffer containing a carrier protein such as 0.1% BSA (bovine serum albumin) or 10% FBS (fetal bovine serum), unless your experiment requires serum-free conditions.
• For serum-free or animal experiments, avoid animal-derived carrier proteins and consider using trehalose as a stabilizer.

4. Storage After Reconstitution

• Short-term (≤1 week): Store at 2–8°C (refrigerator).
• Long-term: Aliquot and store at –20°C to –80°C to avoid repeated freeze-thaw cycles.
• For long-term storage, adding 5–50% glycerol can help stabilize the protein.
• Avoid repeated freeze-thaw cycles, as this can degrade the protein.

5. Usage

• Thaw aliquots on ice before use.
• Use freshly thawed aliquots for each experiment to maintain protein integrity.

Summary Table: Key Steps for Recombinant Human Midkine Reconstitution

Additional Notes:

• Always consult the specific Certificate of Analysis (CoA) or product datasheet for any product-specific recommendations.
• If using for sensitive applications (e.g., animal studies, serum-free culture), ensure all reagents are compatible and free from animal-derived components as needed.

These steps will help ensure optimal solubility, stability, and biological activity of recombinant human Midkine in your cell culture experiments.