Separation and detection of large phosphoproteins using Phos-tag SDS-PAGE
Separation and detection of large phosphoproteins using Phos-tag SDS-PAGE
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DOI：
10.1038/nprot.2009.154
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发表时间：
2009-01-01
期刊：
影响因子：
14.8
通讯作者：
Koike, Tohru
中科院分区：
文献类型：
--
作者：
Kinoshita, Eiji;Kinoshita-Kikuta, Emiko;Koike, Tohru
我们提供了一个标准的磷酸盐亲和SDS-PAGE（Mn 2 +-Phos-tag SDS-PAGE）协议，其中Phos-tag用于分析分子量超过200 kDa的大磷蛋白。先前的方案需要12小时的长电泳时间来分离大蛋白质（类似于150 kDa）的磷酸化同种型。该方案使用0.5%（wt/vol）琼脂糖增强的3%（wt/vol）聚丙烯酰胺凝胶，允许在2小时内分离大于200 kDa的蛋白磷酸化同种型。在随后的免疫印迹中，高分子量蛋白质的磷酸化同种型，如哺乳动物雷帕霉素靶蛋白（289 kDa）、共济失调毛细血管扩张突变激酶（350 kDa）和p53结合蛋白1（213 kDa），可以在改进的Mn 2 +-Phos-标签SDS-PAGE凝胶上清楚地检测到上移的迁移条带。在本方案中，从凝胶制备开始到电泳结束的过程需要约4 h。
We provide a standard phosphate-affinity SDS-PAGE (Mn2+-Phos-tag SDS-PAGE) protocol, in which Phos-tag is used to analyze large phosphoproteins with molecular masses of more than 200 kDa. A previous protocol required a long electrophoresis time of 12 h for separation of phosphoisotypes of large proteins (similar to 150 kDa). This protocol, which uses a 3% (wt/vol) polyacrylamide gel strengthened with 0.5% (wt/vol) agarose, permits the separation of protein phosphoisotypes larger than 200 kDa within 2 h. In subsequent immunoblotting, phosphoisotypes of high-molecular-mass proteins, such as mammalian target of rapamycin (289 kDa), ataxia telangiectasia-mutated kinase (350 kDa) and p53-binding protein 1 (213 kDa), can be clearly detected as up-shifted migration bands on the improved Mn2+-Phos-tag SDS-PAGE gel. The procedure from the beginning of gel preparation to the end of electrophoresis requires about 4 h in this protocol.