Purification of myelin basic protein from bovine brain

Myelin basic protein (MBP) is a commonly used substrate for in vitro determ ination of numerous protein kinase activities. Herein we describe a rapid m ethod for isolating relatively large amounts of MBP from bovine brain with a purity greater than that currently available from commercial sources. Lip ids were first extracted from the CNS tissue by homogenization in sec-butan ol. Washes under neutral and mildly basic conditions were employed to remov e neutral and acidic proteins from the defatted residue. MBP was subsequent ly extracted under acidic conditions and further purified by chromatography on CM Sephadex C-25. Potential contaminating enzyme activities were destro yed by heart treatment. This method typically yields a recovery of 1.0-1.5 mg MBP per gram of starting material with a purity of greater than 95%. The MBP prepared in this manner was suitable for determination of kinase activ ities by both solution and the "in gel" kinase assay systems. (C) 2000 Acad emic Press.