Chevalier D, Allen B G
Institut de Cardiologie de Montréal, Centre de Recherche, 5000 rue Bélanger est, Montréal, Québec, H1T 1C8, Canada.
Protein Expr Purif. 2000 Mar;18(2):229-34. doi: 10.1006/prep.1999.1183.
Myelin basic protein (MBP) is a commonly used substrate for in vitro determination of numerous protein kinase activities. Herein we describe a rapid method for isolating relatively large amounts of MBP from bovine brain with a purity greater than that currently available from commercial sources. Lipids were first extracted from the CNS tissue by homogenization in sec-butanol. Washes under neutral and mildly basic conditions were employed to remove neutral and acidic proteins from the defatted residue. MBP was subsequently extracted under acidic conditions and further purified by chromatography on CM Sephadex C-25. Potential contaminating enzyme activities were destroyed 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 activities by both solution and the "in gel" kinase assay systems.
髓鞘碱性蛋白（MBP）是体外测定多种蛋白激酶活性常用的底物。在此，我们描述一种从牛脑中分离相对大量MBP的快速方法，其纯度高于目前市售产品。首先通过在仲丁醇中匀浆从中枢神经系统组织中提取脂质。采用中性和弱碱性条件下的洗涤步骤，从脱脂残渣中去除中性和酸性蛋白质。随后在酸性条件下提取MBP，并通过CM Sephadex C - 25柱层析进一步纯化。通过加热处理破坏潜在的污染性酶活性。该方法通常每克起始材料可回收1.0 - 1.5毫克MBP，纯度大于95%。以这种方式制备的MBP适用于通过溶液法和“凝胶内”激酶测定系统来测定激酶活性。
