Recombinant Human Dtk

Using Recombinant Human Dtk in research applications provides a reliable, consistent, and biologically relevant source of the Dtk protein, enabling precise studies of its function, signaling pathways, and therapeutic potential in human health and disease.

Key reasons to use Recombinant Human Dtk:

• Functional Studies: Recombinant Dtk allows for controlled in vitro and in vivo experiments to elucidate its role in cellular signaling, immune regulation, and disease mechanisms, particularly as Dtk is a member of the TAM (Tyro3, Axl, Mer) receptor tyrosine kinase family involved in cell survival, proliferation, and immune modulation.
• Assay Development: It serves as a standard or positive control in biochemical assays such as ELISA, western blot, and immunohistochemistry, ensuring reproducibility and accuracy in quantifying Dtk or its ligands.
• Drug Discovery: Recombinant Dtk is essential for high-throughput screening of small molecules or antibodies that modulate its activity, supporting the identification of novel therapeutic candidates for diseases where Dtk signaling is implicated, such as cancer, autoimmune disorders, and thrombosis.
• Protein-Protein Interaction Studies: Using recombinant Dtk enables detailed analysis of its interactions with ligands (e.g., Gas6, Protein S) and downstream signaling partners, which is critical for mapping signaling networks and understanding pathophysiology.
• Structural Biology: Recombinant production yields sufficient quantities of Dtk for crystallography or cryo-EM studies, facilitating structural insights that inform drug design and mechanistic understanding.
• Animal Models and Functional Validation: Recombinant Dtk can be administered to animal models to study its physiological effects, validate therapeutic hypotheses, or model disease states.

Advantages of recombinant proteins in general:

• High purity and batch-to-batch consistency compared to native or serum-derived proteins, reducing experimental variability.
• Human origin ensures biological relevance and reduces cross-species artifacts, especially important for translational research.
• Customizability allows for engineering of tags, mutations, or fusion partners to facilitate detection, purification, or functional studies.

In summary, Recombinant Human Dtk is a critical tool for dissecting Dtk-mediated biology, developing diagnostics, and advancing therapeutic research, offering the precision and reproducibility required for modern biomedical science.

Yes, recombinant human Dtk can be used as a standard for quantification or calibration in ELISA assays, provided it is validated for this purpose and matches the assay’s requirements. Recombinant Dtk is commonly supplied as a standard in commercial ELISA kits designed to quantify Dtk in biological samples.

Key considerations and best practices:

• Parallelism and Validation: The recombinant Dtk standard should exhibit parallel dilution curves with natural Dtk found in biological samples, confirming that the assay quantifies both forms equivalently. This ensures accurate calibration and quantification.

• Standard Curve Preparation: Prepare a standard curve using serial dilutions of the recombinant Dtk, typically covering the assay’s dynamic range (e.g., 40–10,000 pg/mL). The standard curve allows calculation of unknown sample concentrations based on their optical density (OD) readings.

• Assay Calibration: ELISA kits are calibrated against highly purified recombinant Dtk, and the supplied standard is assigned a mass value based on comparison to a master calibrator. This process ensures consistency and reliability in quantification.

• Specificity: The assay should be specific for Dtk, with no cross-reactivity to related proteins (e.g., Axl, Mer, TrkA). This is typically validated during kit development.

• Use Fresh Standards: For optimal accuracy, reconstitute and use the recombinant Dtk standard fresh for each assay, as recommended by kit protocols.

• Application Scope: Recombinant Dtk standards are suitable for quantifying Dtk in various sample types, including plasma, serum, and cell culture supernatants, as long as the assay is validated for those matrices.

Protocol summary:

1. Reconstitute recombinant human Dtk according to the manufacturer’s instructions.
2. Prepare serial dilutions to generate a standard curve.
3. Run the ELISA with standards and samples in parallel.
4. Use the standard curve to interpolate Dtk concentrations in unknown samples.

In summary: Recombinant human Dtk is appropriate as a standard for ELISA quantification and calibration, provided it is validated for your specific assay format and sample type. Always follow the assay protocol and confirm parallelism between recombinant and natural Dtk for accurate results.

Recombinant Human Dtk (also known as Tyro3) has been validated for several applications in published research, primarily in the context of studying receptor tyrosine kinase signaling, hematopoiesis, and cell biology. The main applications supported by published studies and product validation data include:

1. Binding Activity Assays: Recombinant Human Dtk has been used to measure binding interactions with its ligands, such as Protein S and Gas6, in functional ELISA and other binding assays. These assays are critical for investigating receptor-ligand interactions and signaling pathways.

2. Western Blotting: Dtk/Tyro3 antibodies and recombinant proteins have been validated for use in Western blotting to detect and quantify Dtk expression in cell lysates and tissue samples.

3. Direct ELISA: Recombinant Dtk is used in direct ELISA formats to detect and quantify Dtk or its interactions with antibodies or ligands.

4. Flow Cytometry: Antibodies against Dtk/Tyro3 are validated for flow cytometry, enabling the detection and analysis of Dtk expression on the surface of cells.

5. Functional Studies: Recombinant Dtk is used in functional studies to investigate its role in hematopoiesis, cell survival, and signaling pathways, particularly in the context of embryonic blood cell development and regulation.

6. Cell-Based Assays: Recombinant Dtk proteins and antibodies are used in cell-based assays to study receptor activation, downstream signaling, and cellular responses.

These applications are supported by research using recombinant Dtk proteins and antibodies from reputable suppliers, as well as published studies in peer-reviewed journals. The use of recombinant Dtk in these assays helps elucidate its biological functions and potential therapeutic relevance.

To properly reconstitute and prepare Recombinant Human Dtk (TYRO3) protein for cell culture experiments, follow these best practices based on standard protocols and manufacturer recommendations:

Reconstitution Steps:

1. Centrifuge the Vial:
   Briefly centrifuge the lyophilized protein vial at low speed (3000–3500 rpm for 5 minutes) to ensure all powder is at the bottom.

2. Reconstitution Buffer:
   Reconstitute the protein in sterile distilled water or sterile PBS (pH 7.2–7.4), unless otherwise specified in the product datasheet.

   • Typical recommended concentration: 0.1–1.0 mg/mL (check your specific product datasheet for exact guidance).

3. Gentle Mixing:

   • Add the buffer slowly to the vial.
   • Gently swirl or tap the vial; do not vortex or shake vigorously to avoid protein denaturation.
   • Allow the vial to sit at room temperature for 15–30 minutes with gentle agitation to ensure complete dissolution.

Preparation for Cell Culture:

1. Aliquot the Protein:

   • Prepare working aliquots (preferably >20 μL) to minimize freeze-thaw cycles and protein loss due to adsorption.

2. Add Carrier Protein (for long-term storage):

   • For extended storage, add a carrier protein or stabilizer such as:
     • 0.1% BSA (bovine serum albumin)
     • 5–10% FBS (fetal bovine serum)
     • 5% HSA (human serum albumin)
     • 5% Trehalose (especially for serum-free or in vivo applications)
   • This helps maintain protein stability and activity.

3. Storage:

   • Short-term (≤1 week): Store at 2–8°C (refrigerator).
   • Long-term: Aliquot and store at –20°C to –80°C in a manual defrost freezer.
   • Avoid repeated freeze-thaw cycles.

4. Dilution for Cell Culture:

   • Dilute the reconstituted protein in cell culture medium or PBS containing carrier protein (if needed) to the desired working concentration.
   • For serum-free or in vivo experiments, use trehalose as a stabilizer instead of animal-derived proteins.

Additional Tips:

• Always refer to the product datasheet or Certificate of Analysis (CoA) for specific instructions, as formulations may vary between suppliers.
• If the protein does not dissolve completely, gently mix at room temperature for several hours or overnight at 4°C.
• For bioactivity assays (e.g., binding to Gas6), follow the recommended assay buffer and concentration ranges.

Summary:

• Reconstitute: 0.1–1.0 mg/mL in sterile water or PBS, gentle mixing, no vortex.
• Stabilize: Add carrier protein (BSA, FBS, HSA, or trehalose) for long-term storage.
• Aliquot: >20 μL per aliquot, avoid freeze-thaw cycles.
• Store: 2–8°C for short-term, –20°C to –80°C for long-term.
• Dilute: In culture medium or buffer with carrier protein as needed.

Following these steps will help ensure optimal protein activity and stability for your cell culture experiments.