Recombinant Human NCAM-L1

Recombinant Human NCAM-L1 is a valuable tool in research applications focused on neural development, cell adhesion, migration, and synaptic plasticity due to its critical roles in mediating cell-cell and cell-matrix interactions, neurite outgrowth, and signaling pathways relevant to both normal physiology and disease states.

Key scientific reasons to use recombinant NCAM-L1 include:

• Modeling Neural Cell Adhesion and Migration: NCAM-L1 is a member of the immunoglobulin superfamily and is essential for neuron-neuron adhesion, neurite fasciculation, and outgrowth. Recombinant NCAM-L1 can be used to coat culture plates, enhancing cell adhesion in a dose-dependent manner, which is useful for in vitro assays of neural cell migration and neurite extension.

• Studying Signal Transduction and Synaptic Plasticity: NCAM-L1 participates in integrin-dependent cell migration via ERK MAP kinase signaling, contributing to processes such as tumor cell invasiveness and neural development. It also acts as a co-receptor for growth factors (e.g., GDNF), influencing Schwann cell migration and neurite growth through activation of FAK and Fyn kinases.

• Investigating Synapse Formation and Stability: NCAM-L1 regulates synapse stabilization and spine maturation, impacting excitatory transmission and synaptic density. Its polysialylation state modulates adhesive properties and promotes neurite outgrowth, which is relevant for studies on neural regeneration and plasticity.

• Cancer and Disease Models: NCAM-L1 is implicated in cell proliferation, motility, migration, and epithelial-mesenchymal transition (EMT) in cancer models, making it useful for research into tumor biology and metastasis.

• Versatile Experimental Applications: Recombinant NCAM-L1 is suitable for a range of applications, including cell culture, ELISA standards, immunohistochemistry, and functional assays. Carrier-free preparations are available for experiments where additives like BSA may interfere.

• Defined Molecular Properties: Recombinant NCAM-L1 provides a consistent, well-characterized protein source, ensuring reproducibility and reliability in experimental protocols.

In summary, using recombinant human NCAM-L1 enables precise investigation of neural adhesion, migration, signaling, and synaptic mechanisms, supporting research in neurobiology, developmental biology, cancer, and regenerative medicine.

Recombinant Human NCAM-L1 can be used as a standard for quantification or calibration in ELISA assays, provided it is properly validated and matched to the assay system. Recombinant proteins are commonly used as standards in quantitative ELISA protocols, as they allow for the generation of a standard curve against which unknown sample concentrations can be interpolated.

Key considerations for use:

• Assay Compatibility: The recombinant NCAM-L1 must be recognized by the capture and detection antibodies used in your ELISA. Most commercial ELISA kits for NCAM-L1/CD56/NCAM-1 are validated to detect both natural and recombinant forms of the protein.
• Standard Curve Preparation: The standard should be reconstituted and diluted according to the kit or protocol instructions, typically in a matrix similar to your samples (e.g., assay buffer, serum, or plasma).
• Protein Form and Purity: Ensure the recombinant NCAM-L1 is of high purity and matches the relevant isoform or epitope recognized by your assay antibodies. NCAM-1/L1CAM has multiple isoforms due to alternative splicing, so the recombinant standard should correspond to the target detected by your ELISA.
• Carrier Protein: Some recombinant proteins are supplied carrier-free or with BSA. Carrier-free formulations are preferred for ELISA standards to avoid interference with antibody binding or signal generation.
• Validation: It is best practice to validate the recombinant standard in your specific ELISA system, confirming parallelism between the standard curve and endogenous sample dilution curves.

Protocol Outline:

1. Reconstitute recombinant NCAM-L1 according to manufacturer instructions (e.g., in sterile PBS).
2. Prepare serial dilutions to cover the expected assay range (e.g., 0.31–150 ng/mL, depending on kit sensitivity).
3. Run the standard curve in parallel with your samples.
4. Calculate sample concentrations by interpolating from the standard curve.

Limitations:

• If your ELISA kit is designed for a specific isoform or post-translational modification (e.g., polysialylation), ensure your recombinant standard matches these features.
• Always confirm that the recombinant protein is suitable for quantitative calibration in your specific assay by consulting the kit documentation or performing a pilot experiment.

In summary, recombinant Human NCAM-L1 is suitable as a standard for ELISA quantification if it is validated for your assay and matches the target detected by your antibodies.

Recombinant Human NCAM-L1 (L1CAM) has been validated in published research for several key applications, primarily in the context of neural development, cell adhesion, neurite outgrowth, and signaling studies.

Key validated applications include:

• Cell adhesion assays: Recombinant NCAM-L1 has been used to study both homophilic (NCAM-NCAM) and heterophilic (NCAM with other molecules) cell-cell interactions, as well as cell-matrix adhesion, often by coating culture plates with the recombinant protein and measuring cell attachment in a dose-dependent manner.
• Neurite outgrowth assays: The protein has been applied to promote and quantify neurite extension in neuronal cell cultures, demonstrating its role in neurite fasciculation and outgrowth, especially in response to neurotrophic factors such as NGF.
• Signaling pathway analysis: Recombinant NCAM-L1 has been used to investigate its modulation of receptor tyrosine kinases (e.g., TrkA) and downstream signaling events, including TrkA autophosphorylation and the activation of pathways involved in neuronal differentiation and migration.
• Synaptic development and plasticity studies: Research has validated its use in examining pre- and postsynaptic roles, synaptic targeting, and selective synaptic pruning, often in combination with other cell adhesion molecules and guidance cues.
• Cell migration assays: The protein has been used to study integrin-dependent and independent mechanisms of cell migration, particularly in neural and glial cells.
• Immunoassays and ELISA standards: Recombinant NCAM-L1 serves as a standard or capture reagent in immunoassays for quantifying NCAM in biological samples.
• Receptor-ligand interaction studies: It has been used to characterize binding to heparan sulfate proteoglycans, agrin, neurocan, and phosphacan, as well as to study its function as a receptor for certain viruses (e.g., rabies virus).

Additional validated uses:

• Western blotting: Recombinant NCAM-L1 is used as a positive control or antigen in immunoblotting experiments.
• Functional studies in tumor biology: It has been applied to investigate roles in tumor cell proliferation, migration, and as a biomarker in cancer research.

Summary Table of Validated Applications

These applications are supported by multiple peer-reviewed studies and reviews, confirming the broad utility of recombinant human NCAM-L1 in neuroscience, cell biology, and disease research.

To reconstitute and prepare Recombinant Human NCAM-L1 protein for cell culture experiments, follow these best-practice steps:

• Centrifuge the vial briefly before opening to ensure all lyophilized protein is collected at the bottom.
• Reconstitute the protein in sterile PBS (phosphate-buffered saline) or sterile distilled water. The recommended concentration for reconstitution is typically 0.1–1 mg/mL; a common working concentration is 100–200 μg/mL.
• Gently mix the solution by pipetting up and down or swirling. Avoid vigorous vortexing or foaming, which can denature the protein.
• Allow the protein to fully dissolve at room temperature for 10–15 minutes.
• Filter sterilize the solution if sterility is critical for your application, using a 0.2 μm filter.

Storage after reconstitution:

• Store the reconstituted protein at 2–8 °C for short-term use (up to 1 week).
• For long-term storage, aliquot and freeze at –20 °C to –70 °C. Avoid repeated freeze-thaw cycles to maintain protein integrity.

Application in cell culture:

• For coating plates to study cell adhesion, a typical concentration is 25 μg/mL in PBS, adding 100 μL per well, and incubating at 37 °C for 1 hour.
• After coating, wash wells with PBS to remove unbound protein before seeding cells.

Additional notes:

• Always consult the specific product datasheet or Certificate of Analysis for any unique requirements, as buffer composition or concentration may vary by manufacturer or protein construct.
• If the protein is tagged (e.g., Fc, His), ensure compatibility with your downstream assay or cell type.

Summary of key steps:

These guidelines ensure optimal protein activity and reproducibility in cell culture experiments.