Recombinant Rat Neurofilament light polypeptide (Nefl) is produced in E.coli and features a full-length mature protein with an expression region spanning amino acids 2-542. This protein includes an N-terminal 10xHis-tag, which helps with purification and detection. The product achieves a purity greater than 85% as determined by SDS-PAGE and is designed for research use only, offering reliable results in experimental applications.
Neurofilament light polypeptide (Nefl) appears to be an essential component of the neuronal cytoskeleton. It seems to play a critical role in maintaining neuronal integrity and function. As part of the neurofilament protein family, Nefl contributes to the structural framework that supports axonal transport and neuronal signaling. Studying this protein may be pivotal for understanding neuronal development and neurodegenerative processes, which makes it a valuable tool in neuroscience research.
Potential Applications
Note: The applications listed below are based on what we know about this protein's biological functions, published research, and experience from experts in the field. However, we haven't fully tested all of these applications ourselves yet. We'd recommend running some preliminary tests first to make sure they work for your specific research goals.
Based on the provided information, the recombinant rat Nefl is expressed in E. coli, a prokaryotic system that is generally unsuitable for producing properly folded eukaryotic cytoskeletal proteins. Nefl requires precise folding and assembly into helical rod domains for its function in neurofilament formation, and it needs to form correct coiled-coil structures for dimerization. E. coli lacks the eukaryotic chaperones and post-translational modification machinery necessary for proper folding of complex structural proteins. The presence of an N-terminal 10xHis tag may further interfere with the native folding, particularly at the N-terminal region, which is important for neurofilament assembly. While the protein is full-length mature (2-542aa) with >85% purity, the expression system makes it highly likely to be misfolded and unable to assemble into neurofilaments properly. Since activity is unverified, the protein cannot be assumed to be correctly folded or bioactive without experimental validation.
1. Antibody Development and Validation
This application is appropriate. The recombinant Nefl can serve as an effective immunogen for generating antibodies that recognize linear epitopes, even if the protein is misfolded. The His-tag facilitates purification and immobilization for screening assays. However, if Nefl is misfolded, antibodies may not recognize conformational epitopes of native, properly assembled Nefl in neuronal tissues. Validation against endogenous Nefl from rat brain extracts is recommended to ensure physiological relevance.
2. Protein-Protein Interaction Studies
The His-tag enables technical feasibility for pull-down assays, but if Nefl is misfolded (as likely in E. coli), it may not interact physiologically with its true binding partners (e.g., other neurofilament subunits NF-M and NF-H, or cytoskeletal linkers). The coiled-coil domains require precise conformation for specific interactions. Identified interactions could be non-physiological artifacts. This application should only be pursued after confirming proper folding and dimerization capability through biophysical characterization.
3. Biochemical Characterization and In Vitro Assembly Studies
This application is valuable for assessing the protein's properties but requires careful interpretation. Basic biochemical studies (stability, solubility) are feasible, but assembly studies are high-risk without folding validation. If Nefl is misfolded, in vitro filament assembly assays will not reflect physiological neurofilament formation. Techniques like turbidity assays and electron microscopy may yield misleading results if the protein cannot form proper coiled-coil dimers. These studies should include validation of proper secondary structure (e.g., by circular dichroism) before interpreting assembly data.
4. Cross-Species Comparative Studies
This application is problematic without folding validation. If the rat Nefl is misfolded, comparisons with properly folded orthologs from other species will be invalid, as observed differences may arise from folding artifacts rather than true evolutionary variations. This application should be deferred until proper folding and assembly capability are confirmed for all proteins being compared.
Final Recommendation & Action Plan
Given the high probability of misfolding in E. coli for this complex structural protein, we recommend first performing a comprehensive biophysical characterization to assess folding quality. This should include circular dichroism spectroscopy to verify the expected α-helical content characteristic of neurofilament proteins, size-exclusion chromatography with multi-angle light scattering to analyze oligomeric state, and cross-linking studies to assess dimer formation. Antibody development can proceed immediately as the safest application. Protein interaction, assembly, and comparative studies should await proper folding validation. For reliable neurofilament assembly studies, consider using Nefl from eukaryotic expression systems capable of proper folding and post-translational modifications. Always include appropriate controls, such as native neurofilaments from neuronal tissues, when possible.
