Recombinant Human TrkA

Recombinant Human TrkA (Tropomyosin receptor kinase A, also known as NTRK1) is a valuable tool for a wide range of research applications due to its critical role in neurotrophic signaling, cell differentiation, and disease mechanisms. Here are several compelling reasons to use Recombinant Human TrkA in your research:

1. Functional Studies and Signaling Pathway Analysis

TrkA is a receptor tyrosine kinase that mediates the effects of nerve growth factor (NGF), leading to activation of key signaling pathways such as Ras-MAPK and PI3K-Akt. Using recombinant TrkA allows you to:

• Investigate the molecular mechanisms of NGF-induced signaling.
• Study downstream events like cell differentiation, neurite outgrowth, and survival.
• Analyze the impact of TrkA activation or inhibition on cellular responses.

2. Binding and Interaction Assays

Recombinant TrkA is ideal for:

• Studying ligand-receptor interactions (e.g., NGF binding).
• Screening for novel TrkA activators or inhibitors.
• Characterizing the effects of mutations or post-translational modifications on receptor function.

3. Therapeutic Development and Drug Screening

TrkA is a target in several diseases, including:

• Cancer: TrkA fusions are found in various cancers, and inhibitors are being developed for precision medicine.
• Neurodegenerative diseases: TrkA activators are being explored for conditions like Alzheimer's disease.Recombinant TrkA enables:
• High-throughput screening of potential therapeutic compounds.
• Validation of drug efficacy and specificity in kinase and cell-based assays.

4. Standardization and Reproducibility

Recombinant proteins offer:

• High purity and lot-to-lot consistency, which is crucial for reproducible experimental results.
• Defined molecular characteristics, such as specific tags (e.g., His-tag, Fc-tag) for easy detection and purification.

5. Disease Modeling and Mechanistic Studies

TrkA is implicated in:

• Congenital insensitivity to pain, anhidrosis, and self-mutilating behavior.
• Neuroblastoma and other cancers.Using recombinant TrkA helps in:
• Modeling disease states and understanding the functional consequences of TrkA mutations.
• Investigating the role of TrkA in tumor biology and patient prognosis.

6. Versatile Applications

Recombinant TrkA can be used in various experimental formats, including:

• Kinase assays: To measure TrkA activity and inhibition.
• Blocking assays: To study the effects of TrkA blockade on cellular processes.
• ELISA and Western blotting: As a standard or positive control.
• Cell culture studies: To investigate the effects of TrkA on cell behavior.

7. High Quality and Reliability

Many recombinant TrkA products are:

• Produced in mammalian or bacterial expression systems to ensure proper folding and post-translational modifications.
• Validated for bioactivity, purity, and low endotoxin levels, ensuring reliable and reproducible results.

In summary, Recombinant Human TrkA is a versatile and reliable reagent that can significantly enhance your research in neurobiology, cancer, and drug development by providing a standardized and well-characterized tool for functional, mechanistic, and therapeutic studies.

Yes, you can use recombinant human TrkA as a standard for quantification or calibration in ELISA assays, provided it is properly characterized and matches the epitope recognized by your assay antibodies. Recombinant proteins are commonly used as standards in ELISA protocols for quantitative measurement of target analytes.

Key considerations for using recombinant TrkA as an ELISA standard:

• Purity and Characterization: The recombinant TrkA should be highly purified (typically >95% by SDS-PAGE) and its concentration accurately determined. This ensures reliable standard curve generation and quantification.

• Epitope Compatibility: The recombinant TrkA must contain the same epitope(s) recognized by the capture and detection antibodies in your ELISA kit. Most commercial ELISA kits for TrkA are validated to detect both natural and recombinant forms, provided the recombinant protein includes the relevant sequence (e.g., extracellular domain).

• Standard Curve Preparation: Prepare serial dilutions of the recombinant TrkA in the same buffer as your samples to generate a standard curve covering the expected concentration range in your assay. Follow the kit-specific instructions for dilution and handling, as improper preparation can affect accuracy.

• Carrier Proteins: Some protocols recommend using recombinant proteins with carrier proteins (e.g., BSA) to improve stability and reduce adsorption losses, especially at low concentrations.

• Validation: It is best practice to validate the recombinant standard in your specific ELISA system, confirming parallelism between the standard curve and sample dilution curves to ensure accurate quantification.

Limitations and Caveats:

• Some ELISA kits are designed to detect only native TrkA and may not recognize recombinant forms if they lack post-translational modifications or proper folding. Always check your kit’s documentation for compatibility.
• If your recombinant TrkA is tagged (e.g., His-tag), ensure that the tag does not interfere with antibody binding or assay performance.

Summary Table: Recombinant TrkA as ELISA Standard

In conclusion, recombinant human TrkA is suitable as an ELISA standard if it is properly characterized, matches the antibody specificity, and is validated in your assay system.

Recombinant Human TrkA (NTRK1) has been validated for a variety of applications in published research, primarily in the context of functional studies, signaling assays, and therapeutic development. Key applications supported by published literature and product validation include:

• Functional Studies: Recombinant TrkA is used to investigate neurotrophic signaling, neuronal differentiation, and cell survival pathways. It is often employed in assays to study the effects of neurotrophins such as NGF (nerve growth factor) on TrkA activation and downstream signaling (e.g., MAPK/ERK and PI3K/AKT pathways) [2, 3, 4, 6].

• Binding Assays: TrkA proteins are used to study ligand-receptor interactions, including the binding of NGF and other neurotrophins, as well as the effects of small molecule modulators or peptide agonists/antagonists [4, 6].

• Kinase Assays: Recombinant TrkA is validated for use in kinase activity assays, measuring phosphorylation events and downstream signaling cascades following receptor activation [7, 14].

• Cell Proliferation and Survival Assays: TrkA proteins are used to assess the inhibition or promotion of NGF-induced proliferation in cell lines such as TF-1 human erythroleukemic cells, serving as a functional readout for receptor activity [1, 3, 5].

• Therapeutic Development: Recombinant TrkA is used in the development and testing of Trk agonists and antagonists, including low molecular weight modulators and peptide-based therapeutics, for conditions such as dry eye disease and neurotrophic keratitis .

• Western Blotting and Protein Detection: TrkA proteins are used as positive controls or standards in immunoblotting experiments to validate antibody specificity and protein expression [10, 12].

• ELISA and Immunoassays: Recombinant TrkA is used as a standard or capture antigen in ELISA and other immunoassays to quantify receptor levels or ligand binding [9, 15].

• Mass Spectrometry and HPLC: TrkA proteins are suitable for analytical applications such as mass spectrometry and high-performance liquid chromatography for protein characterization and purity assessment .

These applications highlight the versatility of recombinant human TrkA in both basic research and translational studies, particularly in neuroscience, oncology, and drug discovery.

To reconstitute and prepare Recombinant Human TrkA protein for cell culture experiments, dissolve the lyophilized protein in sterile buffer—commonly sterile distilled water or sterile PBS—to achieve a stock concentration suitable for your application, typically between 0.1–0.5 mg/mL. Use gentle mixing and avoid vigorous agitation to prevent protein denaturation.

Step-by-step protocol:

• Warm the vial to room temperature before opening to minimize condensation and ensure accurate reconstitution.
• Add sterile buffer (sterile distilled water or PBS) to the vial. For example, add enough buffer to reach a final concentration of 0.1–0.5 mg/mL, or as specified by your experimental requirements.
• Gently mix by inverting or slow pipetting. Do not vortex or shake vigorously, as this can cause foaming and protein denaturation.
• Allow the protein to dissolve for 15–30 minutes at room temperature with gentle agitation.
• Check for complete dissolution. If undissolved material remains, continue gentle mixing until fully dissolved.
• Aliquot the stock solution to avoid repeated freeze-thaw cycles, which can degrade protein activity.
• Store aliquots at −20°C or −80°C for long-term storage. For short-term use, keep at 2–8°C and use within 24 hours.

Preparation for cell culture:

• Dilute the stock solution in cell culture medium or buffer immediately before use to the desired working concentration (e.g., 0.2–1.2 μg/mL for functional assays).
• Filter sterilize the final working solution if required for sensitive cell culture applications.
• Avoid repeated freeze-thaw cycles to maintain protein integrity and activity.

Additional notes:

• Always consult the specific product datasheet for recommended reconstitution buffer and concentration, as requirements may vary depending on protein formulation and intended use.
• If the protein contains a tag (e.g., His-tag or Fc chimera), ensure compatibility with downstream applications and cell types.

This protocol ensures optimal solubility and biological activity of recombinant TrkA protein for cell culture experiments.