Recombinant Mouse Lefty-1

Recombinant Mouse Lefty-1 is used in research applications primarily to study left-right asymmetry during embryonic development, as well as to investigate its role as a modulator of TGF-β family signaling, particularly as an antagonist of Nodal signaling.

Key scientific reasons to use Recombinant Mouse Lefty-1:

• Modeling Left-Right Determination: Lefty-1 is essential for restricting the expression of lefty-2 and nodal to the left side of the developing mouse embryo, which is critical for proper left-right patterning of visceral organs. Recombinant protein enables controlled studies of these mechanisms in vitro and in vivo.

• Functional Assays: Recombinant Lefty-1 has validated bioactivity, including the ability to induce cell death in specific cell lines (e.g., Mv1Lu mink lung epithelial cells), making it suitable for cell-based assays to dissect signaling pathways.

• Antagonist of Nodal Signaling: Lefty-1 acts as an antagonist to Nodal, a key morphogen in embryogenesis, by potentially competing for receptor binding. This property is valuable for mechanistic studies of TGF-β superfamily signaling and its downstream effects.

• Developmental Biology: The protein is widely used to study organogenesis, heart and lung development, and the molecular basis of congenital asymmetry disorders.

• Comparative Studies: Recombinant Mouse Lefty-1 allows for cross-species comparisons, as Lefty homologs are present in other vertebrates (chick, frog, zebrafish), but with conserved expression patterns and functional roles in left-right patterning.

• Protein Structure and Processing: Recombinant preparations often include both propeptide and mature forms, enabling studies on differential processing, structure-function relationships, and post-translational modifications.

• Cancer and Disease Models: Emerging research suggests roles for LEFTY1 in tumor biology, where it may influence cell proliferation and migration, making recombinant protein useful for preclinical studies.

Typical applications include:

• Cell signaling assays
• Developmental biology models
• Protein-protein interaction studies
• Functional genomics
• Comparative embryology

Using recombinant protein ensures batch-to-batch consistency, defined purity, and the ability to manipulate concentration and exposure in experimental systems, which is critical for reproducible and interpretable results in mechanistic studies.

Yes, you can use Recombinant Mouse Lefty-1 as a standard for quantification or calibration in your ELISA assays, provided it is specifically formulated and validated for this purpose.

Key Points:

• Formulation: Recombinant Mouse Lefty-1 proteins intended for use as ELISA standards are typically lyophilized and may include a carrier protein such as BSA (bovine serum albumin) to stabilize the protein and prevent adsorption to container surfaces. For example, R&D Systems offers Recombinant Mouse Lefty-1 Protein (catalog 994-LF) with BSA, which is recommended for use as an ELISA standard.

• Validation: The recombinant protein should be used according to the manufacturer's instructions for reconstitution and dilution. It is important to generate a standard curve using serial dilutions of the recombinant protein to ensure accurate quantification of your samples.

• Specificity: Ensure that the recombinant protein is compatible with the antibodies used in your ELISA kit. The standard should be recognized by both the capture and detection antibodies to produce a reliable standard curve.

• Limitations: Recombinant protein standards included in ELISA kits are calibrated for use as ELISA standards and may not be suitable for other applications such as bioassays, as they may contain carrier proteins and have not undergone the same sterility testing as bioactive recombinant proteins.

Recommendations:

• Always refer to the product datasheet and follow the manufacturer's guidelines for reconstitution and use.
• Validate the standard curve in your specific assay conditions to ensure accuracy and precision.
• If using a recombinant protein from a different source, confirm compatibility with your ELISA kit and validate the standard curve accordingly.

By following these guidelines, you can effectively use Recombinant Mouse Lefty-1 as a standard for quantification or calibration in your ELISA assays.

Recombinant Mouse Lefty-1 protein has been validated for several applications in published research, primarily focusing on its biological activity and use in mechanistic studies. The main applications include:

• Bioactivity assays: Recombinant Mouse Lefty-1 has been used to measure its ability to modulate cellular responses, such as inhibiting TGF-β1-induced fibroblast-myofibroblast transdifferentiation in rat kidney fibroblasts (NRK-49F cells). This demonstrates its functional activity in cell-based assays.

• Western Blotting (WB): Lefty-1 has been detected and analyzed using Western blotting in studies investigating its expression and effects on signaling pathways.

• ELISA: The protein has been used in ELISA applications to quantify its presence or to study its interactions.

• Immunocytochemistry/Immunofluorescence (ICC/IF): Lefty-1 has been visualized in cells using immunocytochemistry and immunofluorescence techniques.

• Cell signaling studies: Recombinant Mouse Lefty-1 has been applied in studies examining its effects on key signaling pathways, such as Smad3, JNK-3, and BMP-5 in the TGF-β/BMP pathway.

These applications are supported by published research and product datasheets, confirming its utility in both functional and analytical contexts.

To reconstitute and prepare Recombinant Mouse Lefty-1 protein for cell culture experiments, dissolve the lyophilized protein at a concentration of 0.5–2 mg/mL in sterile PBS containing at least 0.1% human or bovine serum albumin (BSA). This stabilizes the protein and minimizes adsorption to surfaces.

Step-by-step protocol:

• Briefly centrifuge the vial before opening to ensure all lyophilized material is at the bottom.
• Add sterile PBS (phosphate-buffered saline) containing ≥0.1% BSA to the vial. The BSA acts as a carrier and stabilizer, especially important for low concentration or sensitive proteins.
• Gently mix by swirling or tapping the vial. Avoid vigorous vortexing, which may denature the protein.
• Allow the protein to dissolve at room temperature for several minutes. If necessary, gently pipette up and down to aid dissolution.
• Aliquot the reconstituted protein to avoid repeated freeze-thaw cycles, which can degrade activity.
• Storage after reconstitution:
  • For short-term use (up to 1 month), store at 2–8°C under sterile conditions.
  • For long-term storage (up to 3 months), store at –20°C to –70°C in a manual defrost freezer.
• Avoid repeated freeze-thaw cycles to preserve protein integrity.

Preparation for cell culture:

• Before adding to cells, dilute the stock solution to the desired working concentration using cell culture medium or PBS with BSA, maintaining sterile technique.
• Filter the final solution through a 0.2 μm sterile filter if sterility is a concern and the protein is compatible with filtration.
• Add the protein to cell culture media as required for your experimental design.

Additional notes:

• If your formulation does not contain BSA, you may reconstitute in sterile PBS alone, but the addition of carrier protein is recommended for stability, especially at low concentrations.
• Always consult the specific product datasheet for lot-specific instructions, as formulations may vary.

Summary Table:

This protocol ensures optimal stability and bioactivity of Recombinant Mouse Lefty-1 for cell culture applications.