A procedure for purifying low-abundance protein components from the brain cytoskeleton-nuclear matrix fraction.
A procedure for purifying low-abundance protein components from the brain cytoskeleton-nuclear matrix fraction.
复制标题
从脑细胞骨架-核基质部分中纯化低丰度蛋白质成分的程序。
DOI：
10.1016/0165-0270(91)90032-u
复制
发表时间：
1991
影响因子：
3
通讯作者：
Egle,PM
中科院分区：
文献类型：
--
作者：
Shelton,KR;Klann,E;Nixon,G;Egle,PM
我们描述了一个制备程序的低丰度蛋白质的细胞核基质馏分从冷冻牛脑。避免在制备细胞核基质级分中的严格离心和洗涤条件，以使核材料的损失最小化。最近描述的水平等电聚焦柱，它容忍可观的降水，使用。与尿素浓度和温度的选择一致，该等电聚焦装置提供了一种新的方法来分离这种复杂的、相对不溶性的蛋白质和其他组分的混合物。此外，已使用加热的十二烷基硫酸钠上胶柱，以消除所需的低丰度蛋白质和更丰富的污染蛋白质之间的相互作用。这些程序共同纯化了特定的低丰度蛋白质，足以通过二维凝胶中的考马斯蓝染色进行检测。该方法是稳健的，可以应用于多个，相对较大的大脑样本（每批150克的粗灰质），因此，他们应该有利于部分肽测序的脑蛋白质的这种操作类。
We describe a preparative procedure for low-abundance proteins of the cytoskeleton-nuclear matrix fraction from frozen bovine brain. Stringent centrifugation and washing conditions in the preparation of the cytoskeleton-nuclear matrix fraction are avoided to minimize loss of nuclear material. A recently described horizontal isoelectric focusing column, which tolerates appreciable precipitation, is used. In concert with selection of urea concentration and temperature, this isoelectric focusing apparatus provides a new approach to the fractionation of this complex, relatively insoluble mixture of proteins and other components. In addition, a heated, sodium dodecyl sulfate-sizing column has been utilized in order to eliminate interactions between the desired low abundance proteins and more abundant contaminating proteins. Together these procedures purify a specific low-abundance protein sufficiently to be detected by Coomassie blue staining in two-dimensional gels. The methods are robust and can be applied to multiple, relatively large brain samples (150 g of crude grey matter per batch); thus they should facilitate partial peptide sequencing for brain proteins of this operational class.