Recombinant Human Pleiotrophin

Recombinant Human Pleiotrophin (PTN) is widely used in research due to its critical roles in neuronal development, neuroprotection, cell growth, and metabolic regulation, making it a valuable tool for studies in neuroscience, regenerative medicine, cancer biology, and metabolic disorders.

Key scientific applications and rationale:

• Neuronal Growth and Differentiation: PTN promotes neurite outgrowth, supports neuronal survival, and enhances differentiation and myelination of oligodendrocytes. These properties are essential for studying neural development, neuroregeneration, and mechanisms underlying neurodegenerative diseases.

• Neuroprotection and Repair: PTN has demonstrated efficacy in models of brain injury, including hypoxic-ischemic injury and traumatic brain injury, by promoting neuronal survival and remyelination. Recombinant PTN can be used to investigate neuroprotective pathways and potential therapeutic strategies for CNS injuries.

• Cell Culture Substrate: Recombinant PTN can serve as an attachment substrate to stimulate neurite outgrowth in primary neuronal cultures, facilitating in vitro studies of neuronal morphology and connectivity.

• Mitogenic Activity: PTN is a potent mitogenic cytokine for fibroblasts, endothelial, and epithelial cells, making it useful for research on cell proliferation, tissue repair, and angiogenesis.

• Cancer Biology: PTN is implicated in tumor growth, metastasis, and angiogenesis. It can be used to study tumor microenvironment interactions, cancer cell signaling, and mechanisms of tumor-induced vasculogenesis.

• Metabolic Regulation: PTN modulates inflammatory conditions related to metabolic disorders, influences insulin sensitivity, and regulates metabolic homeostasis. Recombinant PTN is valuable for exploring CNS-peripheral metabolism crosstalk and metabolic disease mechanisms.

• Biochemical and Mechanistic Studies: PTN interacts with multiple receptors (e.g., PTPRZ, ALK, syndecan-3, nucleolin), enabling mechanistic studies of receptor-mediated signaling pathways in various cell types.

Best practices for use:

• Employ recombinant PTN in defined cell culture systems to study its effects on cell differentiation, survival, and signaling.
• Use in animal models to assess neuroprotective or regenerative outcomes following injury.
• Apply in biochemical assays to dissect receptor interactions and downstream signaling mechanisms.
• Ensure high purity and low endotoxin levels for sensitive applications, such as primary cell culture or in vivo studies.

Summary:
Use recombinant human pleiotrophin in research when investigating neuronal development, neuroprotection, cell proliferation, cancer biology, or metabolic regulation, as it provides a controlled and reproducible means to study PTN’s diverse biological functions and therapeutic potential.

Yes, recombinant human Pleiotrophin can be used as a standard for quantification or calibration in ELISA assays, provided it is properly validated and matched to the assay system. Recombinant proteins are commonly used as standards in ELISA protocols to generate standard curves for quantification of target analytes.

Key considerations for using recombinant human Pleiotrophin as an ELISA standard:

• Assay Compatibility: Many commercial ELISA kits for human Pleiotrophin/PTN are designed to recognize both natural and recombinant forms of the protein, allowing recombinant standards to be used for calibration.
• Standard Curve Preparation: The recombinant protein should be reconstituted and diluted according to the assay protocol to generate a standard curve covering the expected concentration range in your samples.
• Validation: It is essential to confirm that the recombinant standard behaves similarly to the endogenous protein in your assay system. This includes comparable binding to the capture and detection antibodies and similar signal generation.
• Formulation: Recombinant Pleiotrophin is available in carrier-free and carrier-added formulations. Carrier proteins (e.g., BSA) may improve stability but could interfere with certain assays. Choose the formulation that best matches your assay requirements.
• Purity and Activity: Ensure the recombinant protein is of high purity and retains biological activity, as specified by the manufacturer or validated in your lab.

Protocol Best Practices:

• Reconstitute the recombinant standard in the recommended buffer (often PBS with or without carrier protein) and avoid repeated freeze-thaw cycles to maintain protein integrity.
• Prepare serial dilutions to cover the dynamic range of your ELISA.
• Include appropriate controls to verify the accuracy and reproducibility of quantification.

Limitations and Caveats:

• Some ELISA kits are designed specifically for native (endogenous) PTN and may not be validated for recombinant standards. Always check the kit documentation.
• If the recombinant protein is glycosylated or otherwise modified differently than the native protein, this could affect antibody recognition and quantification accuracy.

Summary Table: Recombinant Pleiotrophin as ELISA Standard

In summary: Recombinant human Pleiotrophin is suitable as a standard for ELISA quantification if it is validated for your assay system and matches the kit’s requirements. Always follow best practices for standard preparation and assay validation to ensure accurate quantification.

Recombinant Human Pleiotrophin (PTN) has been validated for a broad range of applications in published research, particularly in studies of neural development, bone biology, cancer, and cell signaling.

Key validated applications include:

• Functional Assays: PTN is widely used to assess its biological activity, such as stimulating neurite outgrowth, promoting cell proliferation, and inducing differentiation in various cell types, including neuronal, osteoblastic, and stem cells.
• Cell Culture: PTN serves as a substrate or supplement to stimulate neurite outgrowth in primary neuronal cultures and to study its effects on cell attachment, proliferation, and differentiation.
• Bioassays: PTN is used in bioassays to evaluate its mitogenic, angiogenic, and chemotactic properties, as well as its effects on osteogenesis, bone regeneration, and tumor cell behavior.
• ELISA: PTN is used as a standard or analyte in ELISA to quantify its levels in biological samples, particularly in cancer and biomarker studies.
• Immunohistochemistry (IHC): PTN is used to validate antibody specificity and to study its tissue distribution in various physiological and pathological contexts.
• Western Blot: PTN is used as a positive control or to validate antibody specificity in protein detection assays.
• Blocking Assays: PTN is used to block or compete for receptor binding in mechanistic studies of its signaling pathways.

Published research examples:

• Neural research: PTN is used to stimulate neurite outgrowth in embryonic brain cell cultures and to study neuronal differentiation and plasticity.
• Bone biology: PTN-loaded nanoparticles have been shown to enhance osteoblast viability, mineralization, and differentiation, supporting its use in bone regeneration and osteoporosis models.
• Cancer research: PTN is studied for its role in tumor growth, metastasis, and as a biomarker in cancers such as prostate cancer and multiple myeloma.
• Stem cell biology: PTN is used to investigate its effects on stem cell proliferation, differentiation, and niche interactions.

Summary Table of Validated Applications

These applications are supported by both product validation data and peer-reviewed research, demonstrating PTN’s utility in diverse experimental systems.

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

1. Preparation Before Reconstitution

• Centrifuge the vial briefly to ensure all lyophilized powder is at the bottom before opening.
• Work in a sterile environment to prevent contamination.

2. Reconstitution

• Buffer choice: The most common and recommended buffer is sterile phosphate-buffered saline (PBS), pH 7.2–7.4. Alternatively, some protocols allow for sterile distilled water, but PBS is preferred for physiological compatibility.
• Concentration: Reconstitute to a stock concentration of 0.1–1.0 mg/mL (100–1000 μg/mL) depending on your experimental needs. A typical starting point is 100 μg/mL in sterile PBS, or 0.2 mg/mL in PBS with 0.1% human serum albumin (HSA) for added stability.
• Carrier protein: If the protein is carrier-free, consider adding a carrier protein (e.g., 0.1% BSA, HSA, or FBS) to prevent adsorption and loss, especially at low concentrations or for long-term storage. For serum-free or animal-free applications, use recombinant HSA or trehalose as a stabilizer.
• Mixing: Gently swirl or invert the vial to dissolve. Do not vortex as this may denature the protein.

3. Incubation

• Allow the vial to sit at room temperature for 15–30 minutes with gentle agitation to ensure complete dissolution.

4. Aliquoting and Storage

• Aliquot the reconstituted protein into single-use volumes to avoid repeated freeze-thaw cycles.
• Short-term storage: 2–8 °C for up to 1 week.
• Long-term storage: -20 °C to -80 °C for up to 3 months. For long-term storage, dilute in buffer containing carrier protein and/or 5–50% glycerol if compatible with your application.
• Avoid repeated freeze-thaw cycles, which can degrade the protein.

5. Working Solution Preparation

• Before adding to cell cultures, dilute the stock solution to the desired working concentration using cell culture medium or PBS containing carrier protein.
• For neurite outgrowth assays, coat plates with 3–8 μg/mL PTN in PBS.
• For direct addition to cultures, typical working concentrations range from 1–100 ng/mL, but optimize based on your specific assay and cell type.

6. Additional Notes

• If using in serum-free or animal-free systems, avoid animal-derived carrier proteins and use recombinant or synthetic alternatives.
• Always consult the Certificate of Analysis (CoA) or product datasheet for any protein-specific instructions, as formulations and recommended buffers may vary.

Summary Table: Key Steps for Recombinant Human PTN Reconstitution

These steps will help ensure protein stability, activity, and reproducibility in your cell culture experiments.