Coomassie brilliant blue stained SDS-PAGE gel of pig pNF-H preparation. First lane shows molecular weight standards of indicated molecular size. Next three lanes show BSA at 10, 5 and 2.5µg respectively. Next three show the indicated volumes of the pNF-H preparation. The pNF-H was isolated from pig spinal cord using Triton/sucrose extraction method of Leung and Liem as far as the 6M urea solubilization and batch ion exchange chromatography on hydroxyapatite step. The material was then fractionated using a phosphate gradient of 10mM to 300mM on DEAE-cellulose in 6M urea at pH=7.5. The pNF-H elutes cleanly at about 50mM phosphate as shown. The pig pNF-H runs on SDS-PAGE with an apparent molecular weight of 220kDa.
The current product data sheet can be downloaded from here. We also supply preparations of the same protein isolated from bovine tissues, see here.
Neurofilaments are the 10 nm or intermediate filament proteins found specifically in neurons, d are composed predominantly of four major proteins called NF-L, NF-M, NF-H and α-internexin (1). NF-H is the neurofilament heavy or high molecular weight polypeptide and runs on SDS-PAGE gels at 200-220kDa in the heavily phosphorylated axonal form.
The molecule has an unusual and interesting region consisting of multiple Lysine-Serine-Proline peptides, about 40 of these in human. These peptide repeats are heavily phosphorylated on the Serine residues in axons. Enzymatic removal of these phosphate groups will increase the SDS-PAGE mobility to about 160kDa, likely due to protein conformational changes due to the removal of charge (2). Even the non-phosphorylated form runs aberrantly on SDS-PAGE, as the real molecular weight of NF-H is about 110kDa, with some variation in different species. This is likely due to an unusually high content of charged amino acids. Non-phosphorylated forms of NF-H are found in dendrites and perikarya and early in development, but the majority of NF-H in the adult is this heavily phosphorylated axonal form. Our preparation was isolated from pig spinal cord using a modification of the method of Leung and Liem (3), which purifies out the heavily phosphorylated axonal form, often referred to as pNF-H. The HGNC name for this protein is NEFH.
The current product data sheet can be downloaded from here. We also supply preparations of the same protein isolated from bovine tissues, see here.
1. Perrot, R., Berges, R., Bocquet, A and Eyer, J. Review of the multiple aspects of neurofilament functions, and their possible contribution to neurodegeneration. Mol. Neurobiol 38:27-65 (2008).
2. Julien, J-P. and Mushynski, W. E. Multiple phosphorylation sites in mammalian neurofilament polypeptides. J. Biol. Chem. 257:10467-10470 (1982).
3. Leung, C. L. and Liem, R. K. H. Isolation of intermediate filaments. Prot. Cell Biol. 3:Unit 3.23 doi: 10.1002/0471143030.cb0323s31 (2006).
