Recombinant Human DAN

Recombinant Human DAN is used in research applications to study its role as a secreted antagonist of bone morphogenetic proteins (BMPs), which are critical regulators of cell differentiation, development, and tissue homeostasis. Using recombinant DAN enables precise investigation of BMP signaling pathways, cellular responses, and potential therapeutic interventions in areas such as cancer, fibrosis, and developmental biology.

Key scientific reasons to use Recombinant Human DAN:

• Defined Activity and Purity: Recombinant proteins provide consistent, batch-to-batch reproducibility and high purity, minimizing experimental variability and ensuring reliable results in functional assays.
• Mechanistic Studies: DAN is a member of the DAN family of BMP antagonists. Recombinant DAN allows researchers to dissect its specific inhibitory effects on BMP signaling, facilitating studies on cell fate decisions, tissue patterning, and disease models where BMPs are implicated.
• Therapeutic Research: BMP antagonists like DAN are being explored for their potential in modulating pathological processes such as tumor progression, fibrosis, and abnormal bone formation. Recombinant DAN is essential for preclinical studies evaluating its therapeutic efficacy and safety.
• Cellular and Molecular Assays: Recombinant DAN can be used in cell culture systems to modulate BMP activity, enabling studies on stem cell differentiation, embryogenesis, and tissue engineering.
• Structural and Biochemical Analysis: Recombinant production allows for the generation of sufficient quantities of DAN for structural studies (e.g., crystallography, binding assays), which are crucial for understanding its interaction with BMPs and other partners.

Best practices for using recombinant DAN:

• Use validated protocols for protein handling and storage to maintain bioactivity.
• Employ appropriate controls (e.g., untreated, BMP-treated) to interpret DAN’s effects in experimental systems.
• Confirm protein identity and purity by SDS-PAGE and mass spectrometry before use in critical assays.

In summary, recombinant Human DAN is a vital tool for elucidating BMP-related signaling mechanisms, developing targeted therapies, and advancing basic and translational research in developmental biology and disease.

Yes, recombinant Human DAN can be used as a standard for quantification or calibration in ELISA assays, provided it is properly characterized and matched to the assay system. Recombinant proteins are commonly used as standards in ELISA to generate a standard curve for quantification, especially when purified native protein is unavailable.

Key considerations for using recombinant Human DAN as an ELISA standard:

• Purity and Characterization: The recombinant DAN should be well-characterized and of high purity. Impurities or incorrect folding can affect assay accuracy.
• Assay Compatibility: The standard must be recognized by the antibodies used in your ELISA. Most commercial ELISA kits for DAN use recombinant Human DAN as the calibrator, and their antibodies are validated against this form.
• Standard Curve Preparation: Prepare serial dilutions of the recombinant DAN in the same buffer/matrix as your samples to minimize matrix effects. Follow the kit or assay protocol for recommended concentration ranges and curve fitting (typically 4-PL logistic regression).
• Validation: If you are developing your own ELISA or modifying an existing kit, validate the recombinant DAN standard for accuracy, precision, recovery, and linearity in your sample types.

Practical Protocol:

• Reconstitute the recombinant DAN according to the supplier’s instructions or your lab’s SOP.
• Prepare a standard curve using serial dilutions, covering the expected range of sample concentrations (e.g., 27.43–20,000 pg/mL for some kits).
• Run the standard curve in parallel with your samples and use it to interpolate sample concentrations.

Limitations and Best Practices:

• If using recombinant DAN outside a validated kit, calibrate it against a mass-calibrated reference standard if available.
• Ensure the recombinant DAN is from the same species and isoform as the target in your samples.
• Avoid extrapolating sample concentrations outside the validated standard curve range, as accuracy cannot be guaranteed.

Summary Table:

In conclusion, recombinant Human DAN is suitable as an ELISA standard if it meets the above criteria and is validated for your specific assay system.

Recombinant Human DAN (also known as DAND1) has been validated in published research primarily for applications in cell signaling studies, developmental biology, and functional assays related to BMP (Bone Morphogenetic Protein) pathway inhibition. However, direct evidence from the provided search results specifically detailing validated applications for recombinant human DAN is limited.

Essential context and supporting details:

• DAN/DAND1 Function: DAN is a secreted glycoprotein and a member of the DAN family of BMP antagonists, which modulate signaling pathways critical for embryonic development, tissue differentiation, and organogenesis. Recombinant forms are used to study these biological processes in vitro and in vivo.

• Typical Validated Applications (based on recombinant protein best practices and DAN’s known biology):

  • In vitro cell-based assays: Recombinant human DAN is commonly used to inhibit BMP signaling in cultured cells, allowing researchers to dissect the role of BMPs in cell differentiation, proliferation, and apoptosis.
  • Developmental biology models: DAN proteins are used in embryonic stem cell differentiation protocols and in animal models to study effects on tissue patterning and organ development.
  • Biochemical and binding assays: Recombinant DAN is used to characterize its interaction with BMP ligands and receptors, often via ELISA, SPR (surface plasmon resonance), or co-immunoprecipitation.

• Related recombinant protein applications: While not specific to DAN, recombinant proteins like DAN are widely validated for:

  • Functional blocking experiments (e.g., neutralizing BMP activity in cell culture).
  • Signal transduction studies (e.g., monitoring downstream SMAD phosphorylation after BMP inhibition).
  • Organoid and 3D culture systems to modulate growth factor environments.

• Published research examples: While the search results do not cite specific studies using recombinant human DAN, the protein’s role as a BMP antagonist is well-established in the literature, and recombinant forms are routinely used for:

  • BMP pathway inhibition in neural, skeletal, and cardiac development studies.
  • Investigating DAN’s role in cancer biology, where BMP signaling is implicated in tumor progression and metastasis.

Summary Table: Typical Validated Applications for Recombinant Human DAN

Limitations:
The search results do not provide direct citations of specific published studies validating recombinant human DAN in these applications. The above applications are inferred from DAN’s known biological function and standard uses of recombinant BMP antagonists in research. For precise validation data, consult primary research articles or product datasheets from scientific literature databases.

If you need references to specific peer-reviewed studies or protocols, please clarify, and I can provide more targeted information.

To reconstitute and prepare Recombinant Human DAN protein for cell culture experiments, dissolve the lyophilized protein in sterile water or buffer to a concentration of 0.1–1.0 mg/mL, then dilute further in cell culture medium or buffer containing a carrier protein such as 0.1% BSA to stabilize the protein and prevent adsorption.

Step-by-step protocol:

• Equilibrate the vial: Allow the lyophilized DAN protein vial to reach room temperature before opening to prevent condensation.
• Add sterile solvent: Add sterile distilled water or a suitable buffer (e.g., PBS, 10 mM Tris-HCl, pH 7.4) to achieve a final concentration of 0.1–1.0 mg/mL, depending on your experimental requirements.
• Gentle mixing: Gently swirl or invert the vial to dissolve the protein. Avoid vigorous shaking or vortexing, which can cause foaming and protein denaturation.
• Incubation: Let the solution stand at room temperature for 15–30 minutes with gentle agitation to ensure complete dissolution.
• Stabilization (optional but recommended): If the protein will be stored or used at low concentrations, dilute the reconstituted protein with a carrier protein solution (e.g., 0.1% BSA in PBS) to minimize adsorption and loss.
• Sterile filtration: If sterility is required, filter the solution through a 0.2 µm sterile filter.
• Aliquot and storage: Aliquot the solution to avoid repeated freeze-thaw cycles and store at –20°C or –80°C for long-term storage.

For cell culture experiments:

• Dilute the reconstituted DAN protein into your cell culture medium immediately before use.
• If using serum-free medium, always include a carrier protein (such as BSA) to stabilize the recombinant protein and prevent loss due to adsorption to plasticware.

Additional notes:

• The optimal working concentration for cell culture should be determined empirically, but typical starting concentrations range from 10–100 ng/mL, depending on the biological activity and assay sensitivity.
• Avoid repeated freeze-thaw cycles, as these can degrade the protein.

This protocol is general for recombinant human proteins and should be adapted if the manufacturer provides specific instructions for the DAN protein. Always consult the product datasheet for any protein-specific recommendations.