Recombinant Mouse Protein TRIQK (Triqk)

What is Recombinant Mouse TrkA protein and what is its significance in neurological research?

Recombinant Mouse TrkA, also known as Neurotrophic tyrosine kinase receptor type 1 (NTRK3), is a member of the nerve growth factor tyrosine kinase receptor family. The protein plays a crucial role in the development and function of the nervous system. The typical recombinant mouse TrkA structure consists of two cysteine-rich domains, a cluster of three leucine-rich motifs, and two immunoglobulin-like domains in the extracellular region, complemented by a single transmembrane domain and an intracellular tyrosine kinase domain . This receptor is essential for the survival of sympathetic neurons in vivo and has significant implications for neurological research, particularly in studying neurotrophin signaling pathways .

Mouse TrkA exhibits high sequence homology with its counterparts in other species, sharing 79% amino acid identity with human TrkA and 93% with rat TrkA . These conservation patterns make mouse TrkA a valuable model for translational research while requiring careful consideration of species-specific differences when extrapolating results.

How should Recombinant Mouse TrkA be reconstituted and stored for optimal stability?

For optimal experimental outcomes, proper handling of Recombinant Mouse TrkA is essential. The lyophilized protein typically comes in a 0.2 μm filtered solution in PBS and should be reconstituted at a concentration of 500 μg/mL in PBS . After reconstitution, it is recommended to:

• Store aliquots at -20°C to -80°C to prevent protein degradation from multiple freeze-thaw cycles

• Use a manual defrost freezer rather than auto-defrost to maintain stable temperature

• Avoid repeated freeze-thaw cycles which can compromise protein integrity and activity

For short-term use (1-2 weeks), reconstituted protein may be stored at 2-8°C, but this should be validated for your specific experimental conditions.

What is the difference between carrier-free (CF) and BSA-containing formulations of Recombinant Mouse TrkA?

The choice between carrier-free (CF) and BSA-containing formulations depends on the specific research application:

How can I verify the biological activity of Recombinant Mouse TrkA in my experimental systems?

Verification of recombinant TrkA activity is crucial before proceeding with complex experiments. A standard assay involves measuring the inhibition of NGF-induced proliferation of TF-1 human erythroleukemic cells. For Recombinant Mouse TrkA Fc Chimera, the reported ED50 for this effect is 4-24 ng/mL when used in the presence of 2 ng/mL Recombinant Mouse beta-NGF .

Verification protocol steps:

• Culture TF-1 cells according to established protocols

• Treat cells with 2 ng/mL Recombinant Mouse beta-NGF

• Add varying concentrations of Recombinant Mouse TrkA (1-100 ng/mL)

• Measure cell proliferation after 48-72 hours using standard assays (MTT, BrdU, etc.)

• Calculate ED50 values and compare to reference ranges

Additionally, protein quality can be assessed via SDS-PAGE under reducing and non-reducing conditions, which should show bands at 100-125 kDa and 200-250 kDa, respectively .

What are the known binding partners for TrkA and their relative affinities?

TrkA exhibits specific binding patterns within the neurotrophin family. Understanding these interactions is essential for designing experiments that accurately model neurotrophin signaling:

Each Trk family member shows distinct ligand specificities: TrkA binds NGF and NT-3, TrkB binds BDNF, NT-3, and NT-4/5, while TrkC exclusively binds NT-3 . These specific interaction patterns contribute to the diverse functions of neurotrophins in nervous system development.

What methodological approaches can be used to study TrkA-mediated signaling pathways?

Several established methodological approaches can be employed to investigate TrkA-mediated signaling:

• Receptor Blockade Studies: Recombinant Mouse TrkA Fc Chimera can be used as a molecular tool to sequester NGF, preventing activation of cellular TrkA receptors. This approach helps identify NGF-dependent signaling pathways .

• Retrograde Transport Analysis: NGF activates retrograde transport of TrkA endosomes for association with actin-modulatory proteins. This process can be tracked using fluorescently labeled proteins or antibodies to study:

  • F-actin disassembly dynamics

  • Maturation into transport-competent signaling endosomes

  • Retrograde axonal transport mechanisms

• Immunoprecipitation Studies: These can identify TrkA-interacting proteins following NGF stimulation, helping to map the complete signaling network.

• Mouse Models: Various knockout or transgenic mouse models have demonstrated that TrkA is essential for the survival of sympathetic neurons in vivo, providing important tools for studying neurotrophin function in complex physiological contexts .

What considerations are important when using recombinant TrkA proteins in mouse models?

When designing mouse model experiments with recombinant TrkA proteins, several factors warrant careful consideration:

• Immunization Protocol: Based on protocols used for similar recombinant proteins, a typical immunization might involve mixing 100 μg of protein with Freund's adjuvant in a 1:2 ratio, followed by subcutaneous injection .

• Immunogenicity Assessment: Since recombinant TrkA often contains fusion tags (e.g., Fc regions), researchers should evaluate potential immune responses against both the target protein and fusion partners.

• Delivery Method: Different administration routes (subcutaneous, intraperitoneal, intrathecal) may yield varied biodistribution and efficacy profiles.

• Dosage Optimization: Effective doses may range from 50-100 μg per mouse based on similar protein studies, but this requires optimization for each specific research question .

• Control Groups: Appropriate controls should include protein vehicle alone, irrelevant recombinant proteins of similar size/structure, and where applicable, native TrkA protein.

How do isoforms and post-translational modifications affect TrkA function?

TrkA exists in multiple isoforms with distinct tissue distributions and functional properties:

Post-translational modifications, particularly phosphorylation of the intracellular tyrosine kinase domain, are critical for TrkA signaling. Research methodologies for studying these modifications include phospho-specific antibodies, mass spectrometry approaches, and in vitro kinase assays.

Understanding these structural variations is essential for correctly interpreting experimental results and developing targeted therapeutic approaches for TrkA-related pathologies.

How is TrkA being explored as a therapeutic target in neurological and non-neurological conditions?

Recent clinical and preclinical studies have identified TrkA as a promising therapeutic target across several disease areas:

• Pain Management: In clinical trials, TrkA inhibitors have demonstrated significant pain improvement in patients with knee osteoarthritis, suggesting a role for TrkA in pain signaling pathways .

• Dermatological Applications: TrkA inhibitors have shown efficacy in reducing psoriatic pruritus, which occurs via a TrkA-dependent mechanism .

• Neurodegeneration: Given TrkA's role in neuronal survival, approaches to enhance TrkA signaling are being explored for neurodegenerative conditions.

Research methodologies in this area typically include:

• Target engagement studies

• Efficacy in disease-specific animal models

• Pharmacokinetic/pharmacodynamic modeling

• Biomarker development for clinical translation

What methodological challenges exist in studying TrkA-neurotrophin interactions?

Several technical challenges must be addressed when investigating TrkA-neurotrophin interactions:

• Receptor Specificity: Given the cross-reactivity between neurotrophins and Trk receptors, experimental designs must carefully control for specificity using receptor-selective ligands or blocking antibodies.

• p75NTR Co-receptor Effects: TrkA signaling is modulated by the p75 neurotrophin receptor (p75NTR), which binds all neurotrophins with low affinity and can synergize or antagonize Trk receptor signaling . Methods to differentiate these pathways include:

  • Selective receptor knockdown/knockout approaches

  • Receptor-specific ligands

  • Domain-specific blocking antibodies

• Signaling Complex Formation: TrkA forms multiprotein signaling complexes that vary by cell type and activation state. Advanced proteomics and proximity labeling approaches can help identify context-specific interaction partners.

• Tissue-Specific Expression: The differential expression of TrkA isoforms between neuronal and non-neuronal tissues requires careful selection of experimental systems that match the biological context being studied .

Addressing these methodological challenges is essential for generating reliable data that can advance our understanding of TrkA biology and its therapeutic potential.