Recombinant Mouse Thrombopoietin (E. Coli Expressed)

Recombinant Mouse Thrombopoietin (TPO) expressed in E. coli is widely used in research applications for its ability to stimulate megakaryocyte proliferation, promote platelet production, and support hematopoietic stem cell expansion in vitro and in vivo.

Key scientific reasons to use this reagent include:

• Megakaryocyte and Platelet Regulation: TPO is the primary physiological regulator of megakaryopoiesis and thrombopoiesis, directly stimulating the proliferation and differentiation of megakaryocytes, which are precursors to platelets.
• Hematopoietic Stem Cell Expansion: TPO promotes the survival, self-renewal, and expansion of hematopoietic stem cells (HSCs) and primitive multilineage progenitors, making it essential for stem cell culture and transplantation studies.
• Functional Assays: Recombinant mouse TPO is used in cell culture to induce megakaryocyte differentiation, support in vitro expansion of HSCs, and activate platelets for functional and apoptosis assays.
• Immunomodulatory Effects: TPO has been shown to modulate immune cell populations, such as increasing CD4+ T cells and restoring immune homeostasis, which is relevant for studies on immune recovery and hematopoietic disorders.
• Consistency and Scalability: E. coli-expressed recombinant proteins offer batch-to-batch consistency, are free from animal-derived contaminants, and are suitable for large-scale experimental use.

Typical applications include:

• In vitro megakaryocyte differentiation and proliferation assays.
• Expansion and maintenance of hematopoietic stem and progenitor cells.
• Platelet activation and apoptosis studies.
• Modeling thrombocytopenia and bone marrow recovery in murine systems.
• Investigating immune modulation and hematopoietic lineage recovery.

Technical considerations:

• E. coli-expressed TPO is typically non-glycosylated, which may affect activity or stability compared to mammalian-expressed forms, but it remains highly effective for most cell-based and biochemical assays.
• Biological activity is confirmed by its ability to stimulate cell proliferation in relevant cell lines (e.g., MO7e human megakaryocytic leukemic cells).

In summary, recombinant mouse TPO (E. coli expressed) is a robust tool for studying megakaryopoiesis, thrombopoiesis, stem cell biology, and immune modulation in mouse models and cell culture systems.

Yes, you can use recombinant mouse Thrombopoietin (TPO) expressed in E. coli as a standard for quantification or calibration in your ELISA assays, provided that the recombinant protein is purified and its concentration is accurately determined.

Key Considerations:

• Purification and Concentration: The recombinant protein should be purified to homogeneity, and its concentration should be measured using a reliable method such as amino acid analysis, quantitative Western blot, or spectrophotometry (e.g., absorbance at 280 nm with an extinction coefficient appropriate for the protein).

• Immunoreactivity: The recombinant TPO must be recognized by the antibodies used in your ELISA kit. Most commercial ELISA kits are validated for both natural and recombinant TPO, but it is important to confirm that the epitopes recognized by the capture and detection antibodies are present in your recombinant protein.

• Matrix Effects: The standard should ideally be diluted in a matrix similar to your samples (e.g., assay diluent or buffer that mimics the sample matrix) to minimize matrix effects.

• Validation: It is recommended to validate your standard by comparing it to a commercially available, kit-provided standard if possible, to ensure accurate quantification.

Additional Notes:

• Some ELISA kits specifically recommend using their provided standard, which is often recombinant TPO produced in a mammalian system. However, E. coli-expressed recombinant TPO is commonly used as a standard in research ELISAs, especially when the protein is properly purified and characterized.

• If you are using a commercial ELISA kit, always refer to the manufacturer’s instructions for guidance on standard preparation and use.

In summary, yes, recombinant mouse TPO expressed in E. coli can be used as a standard for ELISA quantification, as long as it is purified, accurately quantified, and immunoreactive with your ELISA antibodies.

Recombinant Mouse Thrombopoietin (E. coli expressed) has been validated in published research for several key applications, primarily in studies of hematopoiesis and platelet biology.

Validated Applications:

• Functional Assays: Used to assess biological activity, such as stimulation of megakaryocyte proliferation, differentiation, and platelet production in vitro and in vivo.
• Cell Culture: Supports the expansion and maintenance of hematopoietic stem cells and megakaryocyte lineage cells in culture systems.
• Megakaryocyte Proliferation and Differentiation: Directly stimulates the proliferation and maturation of megakaryocytes, the precursor cells for platelets.
• Platelet Production and Activation: Used in models to increase platelet counts, both in vitro and in animal studies, including recovery from thrombocytopenia.
• Apoptosis Assays: Investigates the anti-apoptotic effects of thrombopoietin on hematopoietic cells and its pro-apoptotic effects in neural tissues.
• ELISA and Western Blot: Validated as a standard or control protein in immunoassays for detection and quantification of thrombopoietin or related signaling molecules.
• Stem Cell Expansion: Facilitates ex vivo expansion of pluripotent stem cells for research into regenerative medicine and transplantation.
• Radioprotection and Chemotherapy Recovery: Used in animal models to reduce radiation- and chemotherapy-induced thrombocytopenia and enhance platelet recovery after bone marrow transplantation.

Supporting Details:

• The protein is routinely tested for biological activity in appropriate bioassays, confirming its ability to activate the c-Mpl receptor and downstream signaling pathways relevant to megakaryopoiesis.
• Research-grade recombinant mouse thrombopoietin is sterile-filtered, with high purity and low endotoxin levels, making it suitable for sensitive cell culture and functional studies.
• Published animal studies demonstrate its efficacy in increasing peripheral platelet numbers, supporting its use in thrombocytopenia models and therapeutic research.

Summary Table:

These applications are well-supported in the literature and product validation data, making recombinant mouse thrombopoietin (E. coli expressed) a standard tool for research in hematopoiesis, stem cell biology, and platelet function.

To reconstitute and prepare Recombinant Mouse Thrombopoietin (E. coli Expressed) for cell culture experiments, briefly centrifuge the vial to collect the lyophilized protein at the bottom, then reconstitute in sterile distilled water or buffer to a concentration of 0.1–1.0 mg/mL.

Step-by-step protocol:

• Centrifuge the vial briefly before opening to ensure all contents are at the bottom.
• Add sterile distilled water (or sterile PBS) to achieve a final concentration between 0.1–1.0 mg/mL. For example, add 100 µL water to 100 µg protein for 1 mg/mL.
• Gently mix by pipetting up and down or swirling. Avoid vigorous vortexing to prevent protein denaturation.
• Allow several minutes for complete dissolution. Inspect visually to confirm the protein is fully dissolved.
• Aliquot the reconstituted solution into sterile tubes to avoid repeated freeze-thaw cycles.
• Store aliquots at ≤ –20°C for long-term storage. For short-term use, store at 2–8°C for up to one month.
• For working dilutions, further dilute the stock in cell culture medium or buffer containing a carrier protein such as 0.1% heat-inactivated FCS (fetal calf serum) or tissue culture grade BSA (bovine serum albumin) to minimize adsorption and loss.
• Endotoxin levels are typically <0.1 ng/µg, suitable for cell culture applications.
• Determine optimal working concentration for your assay by performing a dose-response experiment, as biological activity may vary depending on cell type and experimental conditions.

Additional notes:

• Use low endotoxin reagents and sterile technique throughout preparation to avoid contamination.
• Avoid repeated freeze-thaw cycles, as this can reduce protein activity.
• If the protein is difficult to dissolve, gentle warming (room temperature) or brief sonication may help, but avoid excessive heat.

This protocol is suitable for most cell culture applications involving recombinant mouse thrombopoietin expressed in E. coli. Always consult the specific product datasheet for additional details relevant to your batch.