Isolation, purification myelin basic protein (MBP) and generation specific antibodies against its

Myelin basic proteins (MBP) are extraneous membrane proteins that play an essential role in the original organization of the myelin sheath. The classic (MBP isoforms are inherently disordered proteins of 14-21.5 kD gain size emerging from the Golli Gene in the Oligodendrocyte Lineage) gene combination, and are accountable for configuration of the multilayered myelin sheath in the central nervous system. The 'classic' 18.5-kDa isoform of myelin basic protein (MBP) was primary isolated more than 50 years ago as an encephalitogenic determinant. This protein is an integral part of central nervous system (CNS) myelin, adhering the cytoplasmic leaflets of the oligodendrocyte (OLG) layer to each other to form the major dense line observed in electron micrographs. This essential role is manifestation by mutant mouse which has an ablation in the MBP gene, and whose CNS myelin is rare and relatively unstructured. In this study MBP was isolated from rat brain 600 mg brain tissue and homogenized. Buffer for homogenates contain the cock tail of the inhibitors proteases benzamidine, leupeptine and PMSF. Rat brain homogenate was centrifuged for a separation of water-soluble proteins. The rat brain homogenate centrifuged for 14000 rpm 60 min and after the pellet collect. This pellet mixed and resuspend with methanol-aceton solution for delipidation. Later 45 min incubation in +4 C0 this mix centrifuged for 30 min 14000 rpm and after the pellet collect. Obtained pellet was used for extraction MBP with acid buffer pH 2.0. The extraction was performed 60 min at +4 C0 with permanent mixing. After centrifugation for 30 min 14000 rpm this extract was used for isolation and purification MBP by SDS/PAAG. Extracted from gel polypeptide with Mm 18 kDa was used as antigen for rabbit immunization. The titer of antibodies in western blot was determinate as maximum dilution for visible results without decreasing intensity of main MBP polypeptides staining on a membrane. This maximal dilution for anti-MBP antibodies was 1:5500. Purification of IgG fraction resulted to obtain specific antibodies against pure MBP from rat brain containing mainly the 18.5 KDa isoform. Obtained specific antibodies against MBP may be successfully used for study demyelinated diseases and olygodendrocyte pathology.