Electroblotting onto activated glass. High efficiency preparation of proteins from analytical sodium dodecyl sulfate-polyacrylamide gels for direct sequence analysis.
Electroblotting onto activated glass. High efficiency preparation of proteins from analytical sodium dodecyl sulfate-polyacrylamide gels for direct sequence analysis.
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电印迹到活性玻璃上。
DOI：
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发表时间：
1986
影响因子：
4.8
通讯作者：
S. Kent
中科院分区：
文献类型：
--
作者：
R. Aebersold;D. Teplow;L. Hood;S. Kent
我们开发了一种分离蛋白质以进行微测序的新方法。它包括在通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳分离后立即将蛋白质或其裂解片段电泳转移（电印迹）到活化的玻璃滤纸上。蛋白质通过离子相互作用或共价连接固定在玻璃纤维片上。可以用这种方式制备多种蛋白质，而不受蛋白质的溶解度、大小、电荷或其他固有特性的明显限制。使用考马斯蓝或荧光染料染色程序可以检测到少至 50 ng 的转移蛋白，甚至可以通过放射自显影检测更少量的放射性标记蛋白。检测后，将含有蛋白质的条带或斑点切下并直接插入气相测序仪中。这片玻璃纤维片在测序过程中充当蛋白质的支撑物。可以对 5 至 150 pmol 范围内的蛋白质含量进行测序，并且由于改进的逐步产量和较低的背景，可以从印迹样品中获得扩展运行。该方法已成功应用于从一维和二维聚丙烯酰胺凝胶中分离的多种蛋白质和肽的测序。
We have developed a new method for the isolation of proteins for microsequencing. It consists of electrophoretic transfer (electroblotting) of proteins or their cleavage fragments onto activated glass filter paper sheets immediately after separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The proteins are immobilized on the glass fiber sheets by ionic interactions or by covalent attachment. A wide range of proteins can be prepared in this fashion with no apparent restriction due to solubility, size, charge, or other intrinsic properties of the proteins. As little as 50 ng of the transferred proteins can be detected using Coomassie Blue or fluorescent dye staining procedures and even smaller amounts of radiolabeled proteins by autoradiography. After detection, the protein-containing bands or spots are cut out and inserted directly into a gas-phase sequenator. The piece of glass fiber sheet acts as a support for the protein during the sequencing. Amounts of protein in the 5- to 150-pmol range can be sequenced, and extended runs can be obtained from the blotted samples because of improved stepwise yields and lower backgrounds. The method has been successfully applied to the sequencing of a variety of proteins and peptides isolated from one-dimensional and two-dimensional polyacrylamide gels.