ELUTION OF PROTEINS FROM SODIUM DODECYL SULFATE-POLYACRYLAMIDE GELS, REMOVAL OF SODIUM DODECYL-SULFATE, AND RENATURATION OF ENZYMATIC-ACTIVITY - RESULTS WITH SIGMA SUBUNIT OF ESCHERICHIA-COLI RNA-POLYMERASE, WHEAT-GERM DNA TOPOISOMERASE, AND OTHER ENZYMES
ELUTION OF PROTEINS FROM SODIUM DODECYL SULFATE-POLYACRYLAMIDE GELS, REMOVAL OF SODIUM DODECYL-SULFATE, AND RENATURATION OF ENZYMATIC-ACTIVITY - RESULTS WITH SIGMA SUBUNIT OF ESCHERICHIA-COLI RNA-POLYMERASE, WHEAT-GERM DNA TOPOISOMERASE, AND OTHER ENZYMES
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DOI：
10.1016/0003-2697(80)90013-5
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发表时间：
1980-01-01
影响因子：
2.9
通讯作者：
BURGESS, RR
中科院分区：
文献类型：
--
作者：
HAGER, DA;BURGESS, RR
本文介绍了一种改进的从十二烷基硫酸钠（SDS）-聚丙烯酰胺凝胶中复性微克量蛋白质的方法。通过KCl染色在凝胶中观察蛋白条带，切下条带并压碎，通过在含有0.1%SDS的缓冲液中扩散洗脱蛋白。浓缩蛋白质，通过丙酮沉淀样品除去SDS。将沉淀物溶于6 M盐酸胍中，然后稀释后，蛋白质发生复性。σ的活性在SDS-凝胶中电泳和复性后，大肠杆菌RNA聚合酶的亚基可以98-100%的效率回收。为了评估该方法是否普遍适用，使用E.大肠杆菌转录终止因子ρ，β-半乳糖苷酶，[细菌]碱性磷酸酶，小麦α-淀粉酶和DNA拓扑异构酶。该方法可用于确定DNA拓扑异构酶多肽的近似分子量，通过将凝胶切片，在凝胶上部分纯的样品已通过电泳分级分离。
An improved method is described for the renaturation of microgram amounts of proteins from sodium dodecyl sulfate[SDS]-polyacrylamide gels. The protein band is visualized in the gel by KCl staining, the band cut out and crushed, and the protein eluted by diffusion in a buffer containing 0.1% SDS. Protein is concentrated and SDS is removed by acetone precipitation of the sample. Renaturation of the protein occurs after the precipitate is dissolved in 6 M guanidine hydrochloride and then diluted. The activity of the .sigma. subunit of Escherichia coli RNA polymerase can be recovered with 98-100% efficiency after electrophoresis in an SDS-gel and renaturation by this technique. To assess whether the method is generally applicable, some or all of the steps involved in the procedure were tested using E. coli transcription termination factor .rho., .beta.-galactosidase, [bacterial] alkaline phosphatase, wheat .alpha.-amylase and DNA topoisomerase. The method can be used to determine the approximate MW of the DNA topoisomerase polypeptide by sectioning a gel on which a partially pure sample has been fractionated by electrophoresis.