Recombinant Human TrkB

Recombinant Human TrkB is used in research applications to study and manipulate the BDNF/TrkB signaling pathway, which is crucial for neuronal survival, differentiation, synaptic plasticity, and regeneration. This protein enables precise, reproducible activation or inhibition of TrkB-mediated cellular processes in vitro and in vivo, facilitating mechanistic studies and therapeutic development.

Key scientific applications and advantages include:

• Modeling Neurotrophic Signaling: Recombinant TrkB allows researchers to dissect the molecular mechanisms of BDNF/TrkB signaling, which is essential for neuronal development, myelination, and regeneration. This is particularly relevant for studies on neurodegenerative diseases, traumatic injury, and psychiatric disorders.

• Screening and Validation of Agonists/Antagonists: Recombinant TrkB is used to test the efficacy and specificity of TrkB agonists (such as BDNF mimetics, small molecules, or agonist antibodies) and antagonists, enabling high-throughput screening and optimization of therapeutic candidates.

• Functional Assays: It serves as a tool in cell-based assays to measure downstream signaling events (e.g., phosphorylation of TrkB, activation of MAPK/Erk and AKT pathways), gene expression changes, and cellular outcomes such as proliferation, differentiation, or apoptosis.

• Disease Modeling and Drug Development: Recombinant TrkB is valuable for modeling diseases where TrkB signaling is dysregulated (e.g., Alzheimer's, multiple sclerosis, cancer), and for evaluating the therapeutic potential of TrkB-targeted interventions.

• Neutralization and Decoy Studies: TrkB-Fc chimeras or soluble recombinant TrkB can act as decoy receptors to sequester BDNF, allowing investigation of TrkB-dependent versus independent effects and the development of neutralizing strategies.

Best practices for use:

• Employ recombinant TrkB in controlled in vitro systems (e.g., reporter cell lines, primary neurons) to ensure specificity of observed effects.
• Validate recombinant protein activity by assessing phosphorylation and downstream signaling responses.
• Use appropriate controls, such as inactive mutants or Fc-only chimeras, to distinguish TrkB-specific effects.

In summary, Recombinant Human TrkB is a versatile and essential reagent for elucidating neurotrophic signaling mechanisms, screening therapeutic candidates, and modeling disease processes involving TrkB.

Yes, recombinant Human TrkB can be used as a standard for quantification or calibration in ELISA assays, provided it is properly characterized and compatible with your assay system. Recombinant TrkB is commonly used as a standard in commercial TrkB ELISA kits, where it is serially diluted to generate a standard curve for quantifying TrkB in biological samples.

Key considerations and best practices:

• Purity and Characterization: The recombinant TrkB standard should be highly purified and its concentration accurately determined, typically by methods such as HPLC or UV absorbance.
• Assay Compatibility: Ensure the recombinant TrkB standard is recognized by the capture and detection antibodies in your ELISA. Most commercial kits use recombinant TrkB as the standard and validate its reactivity.
• Standard Curve Preparation: Prepare serial dilutions of the recombinant TrkB to cover the expected concentration range in your samples (e.g., 0.156–10 ng/mL or up to 20 ng/mL, depending on kit sensitivity).
• Matrix Effects: If quantifying TrkB in complex matrices (e.g., serum, plasma, cell lysates), confirm recovery and linearity by spiking known amounts of recombinant TrkB into these matrices.
• Documentation: Always run a standard curve with each assay and interpolate sample concentrations from this curve.

Limitations:

• The recombinant standard should closely mimic the native protein in your samples in terms of epitope presentation and post-translational modifications. Some ELISA kits note that detection of recombinant protein may differ from native protein due to such differences.
• For absolute quantification, ensure the recombinant TrkB standard is from a reliable source and its concentration is traceable to a reference method.

Summary Table: Use of Recombinant Human TrkB as ELISA Standard

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

Recombinant Human TrkB has been validated for several key applications in published research, primarily in the fields of neurobiology, cell signaling, and therapeutic development.

Validated Applications:

• Cell-based Reporter Assays: Recombinant Human TrkB has been used to generate reporter cell lines (e.g., HEK293 or CHO cells expressing TrkB with CRE or NFAT reporter constructs) for high-throughput screening of TrkB agonists, including antibodies and small molecules. These assays measure TrkB activation, downstream signaling, and transcriptional regulation in response to agonists.

• Kinase Activity Assays: The active recombinant TrkB protein (typically His-tagged, amino acids 455-end) is widely used in in vitro kinase assays to study TrkB autophosphorylation and downstream signaling events.

• ELISA Standards: Recombinant TrkB is used as a standard in ELISA assays to quantify TrkB protein levels in biological samples.

• Cell Proliferation and Functional Assays: TrkB Fc chimera proteins have been used to inhibit BDNF-induced cell proliferation in TrkB-transfected cell lines, demonstrating functional antagonism or modulation of TrkB signaling.

• Gene Therapy and Neuroprotection Studies: Human TrkB has been co-expressed with BDNF in bicistronic AAV vectors for in vivo studies of neuroprotection, particularly in models of retinal injury and glaucoma.

• Western Blot and Immunodetection: Recombinant TrkB is used as a positive control or antigen in Western blotting and immunodetection protocols to validate antibody specificity and TrkB expression.

• Flow Cytometry: Recombinant TrkB protein and TrkB-specific antibodies have been used for flow cytometric analysis of TrkB expression on live cells.

• Drug Screening and Mechanistic Studies: Recombinant TrkB is employed in screening for novel agonists or antagonists, including monoclonal antibodies and small molecules, and in mechanistic studies of TrkB-mediated neurotrophic signaling.

Disease Models and Therapeutic Validation:

• Neurodegenerative Disease Models: Recombinant TrkB and TrkB agonist antibodies have been validated in in vitro and in vivo models of Huntington’s disease, Alzheimer’s disease, and other neurodegenerative conditions to assess neuroprotective effects and downstream signaling.

• Retinal and CNS Injury Models: TrkB-based gene therapy and protein administration have been tested for neuroprotection in models of retinal degeneration and CNS neuroblastoma.

Summary Table of Applications

These applications demonstrate the versatility of recombinant human TrkB in both basic research and translational studies, particularly for investigating neurotrophic signaling, screening therapeutic candidates, and validating disease models.

To reconstitute and prepare Recombinant Human TrkB protein for cell culture experiments, dissolve the lyophilized protein in sterile phosphate-buffered saline (PBS) or sterile distilled water to a concentration typically between 100–500 μg/mL, depending on the specific formulation and experimental requirements.

Step-by-step protocol:

1. Centrifuge the vial briefly to ensure all lyophilized powder is at the bottom before opening.
2. Reconstitution buffer:
   • If the datasheet specifies, use sterile PBS (pH 7.2–7.4).
   • If not specified, sterile distilled water is generally acceptable.
3. Concentration:
   • For most TrkB preparations, reconstitute to 100–500 μg/mL.
   • Avoid concentrations below 100 μg/mL to maintain protein stability.
4. Mix gently:
   • Do not vortex or pipette vigorously; gently swirl or invert to dissolve.
5. Aliquot immediately:
   • Divide the solution into small aliquots to avoid repeated freeze-thaw cycles.
6. Storage:
   • Lyophilized protein: Store at –20°C or colder.
   • Reconstituted solution:
     • Short-term: 4–8°C for 2–7 days.
     • Long-term: –20°C or colder for up to 3 months.
7. Sterility:
   • Use sterile technique throughout to prevent contamination, especially for cell culture applications.

Additional notes for cell culture:

• If using a carrier-free formulation, ensure your buffer does not contain BSA or other proteins that may interfere with downstream assays.
• For sensitive cell types, confirm the buffer composition matches physiological conditions (e.g., isotonicity, pH 7.2–7.4).
• Always consult the specific product datasheet for any unique requirements or recommended buffers.

Summary Table:

Key technical points:

• Use sterile technique throughout.
• Avoid repeated freeze-thaw cycles by aliquoting.
• Confirm buffer and concentration with the product datasheet for optimal activity and stability.

If your experiment requires a specific buffer or additive (e.g., for enhanced stability or activity), refer to the manufacturer’s datasheet for precise recommendations.