Niepmann Michael, Zheng Junfeng
Institute of Biochemistry, Justus-Liebig-University Giessen, Giessen, Germany.
Electrophoresis. 2006 Oct;27(20):3949-51. doi: 10.1002/elps.200600172.
Analysis of the oligomeric state of a native protein usually requires analytical ultracentrifugation or repeated gel filtration to calculate the protein's size. We have developed a discontinuous native protein gel electrophoresis system that allows the separation of even basic proteins according to their size, oligomeric state, and shape. This gel system combines the addition of negative charges to the proteins by Serva Blue G with a discontinuous buffer system and gradient gels. As in SDS-PAGE, chloride constitutes the high mobility anion in the gel and anode buffer. However, for sample focusing this system employs histidine instead of glycine as the slow dipolar ion following from the cathode buffer to improve migration of basic proteins. In addition, proteins run into gel pores corresponding to their size and shape in the gradient gel. Using this gel system, we show that the polypyrimidine tract-binding protein (PTB) is a monomer.
对天然蛋白质的寡聚状态进行分析通常需要使用分析超速离心或反复进行凝胶过滤来计算蛋白质的大小。我们开发了一种不连续的天然蛋白质凝胶电泳系统，该系统能够根据蛋白质的大小、寡聚状态和形状，甚至分离出碱性蛋白质。这种凝胶系统将通过Serva Blue G给蛋白质添加负电荷与不连续缓冲系统和梯度凝胶相结合。与SDS-PAGE一样，氯离子在凝胶和阳极缓冲液中构成高迁移率阴离子。然而，对于样品聚焦，该系统使用组氨酸而非甘氨酸作为从阴极缓冲液流出的慢偶极离子，以改善碱性蛋白质的迁移。此外，蛋白质在梯度凝胶中会进入与其大小和形状相对应的凝胶孔中。使用这种凝胶系统，我们证明了多嘧啶序列结合蛋白（PTB）是一种单体。
