Native SDS-PAGE: high resolution electrophoretic separation of proteins with retention of native properties including bound metal ions.
Native SDS-PAGE: high resolution electrophoretic separation of proteins with retention of native properties including bound metal ions.
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DOI：
10.1039/c4mt00033a
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发表时间：
2014-05
期刊：
影响因子：
--
通讯作者：
Petering DH
中科院分区：
文献类型：
--
作者：
Nowakowski AB;Wobig WJ;Petering DH
十二烷基硫酸钠聚丙烯酰胺凝胶电泳（SDS-PAGE）通常用于获得蛋白质的复杂混合物的高分辨率分离。该方法首先使将经历电泳的蛋白质变性。虽然可以用SDS-PAGE确定解析蛋白质的共价结构特征，但功能特性被破坏，包括非共价结合的金属离子的存在。为了解决这个缺点，已经引入了蓝色-天然（BN）-PAGE。这种方法保留了功能特性，但以蛋白质分辨能力为代价。为了解决对导致天然蛋白质分离的高分辨率PAGE方法的需求，实验测试了改变SDS-PAGE条件对蛋白质分离质量和功能特性保留的影响。从样品缓冲液中去除SDS和EDTA以及省略加热步骤对PAGE结果没有影响。与SDS-PAGE方法相比，将运行缓冲液中的SDS从0.1%降低至0.0375%以及删除EDTA对猪肾（LLC-PK 1）细胞蛋白质组组分的电泳图质量也几乎没有影响。改良的条件称为非变性（N）SDS-PAGE。保留锌结合在蛋白质组样品增加了从26到98%，从标准转换到修改后的条件。此外，九个模型酶中的七个，包括四个进行NSDS-PAGE的Zn 2+蛋白，保留了活性。所有九个在BN-PAGE中是有活性的，而所有在SDS-PAGE期间经历变性。电泳后的金属保留另外确认使用激光烧蚀-电感耦合等离子体质谱法和凝胶锌蛋白染色使用荧光团TSQ。
Sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE) is commonly used to obtain high resolution separation of complex mixtures of proteins. The method initially denatures the proteins that will undergo electrophoresis. Although covalent structural features of resolved proteins can be determined with SDS-PAGE, functional properties are destroyed, including the presence of non-covalently bound metal ions. To address this shortcoming, blue-native (BN)-PAGE has been introduced. This method retains functional properties but at the cost of protein resolving power. To address the need for a high resolution PAGE method that results in the separation of native proteins, experiments tested the impact of changing the conditions of SDS-PAGE on the quality of protein separation and retention of functional properties. Removal of SDS and EDTA from the sample buffer together with omission of a heating step had no effect on the results of PAGE. Reduction of SDS in the running buffer from 0.1% to 0.0375% together with deletion of EDTA also made little impact on the quality of the electrophoretograms of fractions of pig kidney (LLC-PK1) cell proteome in comparison with that achieved with the SDS-PAGE method. The modified conditions were called native (N)SDS-PAGE. Retention of Zn2+ bound in proteomic samples increased from 26 to 98% upon shifting from standard to modified conditions. Moreover, seven of nine model enzymes, including four Zn2+ proteins that were subjected to NSDS-PAGE retained activity. All nine were active in BN-PAGE, whereas all underwent denaturation during SDS-PAGE. Metal retention after electrophoresis was additionally confirmed using laser ablation-inductively coupled plasma-mass spectrometry and in-gel Zn-protein staining using the fluorophore TSQ.