Phos-tag diagonal electrophoresis precisely detects the mobility change of phosphoproteins in Phos-tag SDS-PAGE.
Phos-tag diagonal electrophoresis precisely detects the mobility change of phosphoproteins in Phos-tag SDS-PAGE.
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DOI：
10.1016/j.jprot.2020.104005
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发表时间：
2020-10
影响因子：
3.3
通讯作者：
Yuki Okawara;H. Hirano;Ayuko Kimura;N. Sato;Yuriko Hayashi;M. Osada;T. Kawakami;Norihisa Ootake;E. Kinoshita;Kiyotaka Fujita
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文献类型：
--
作者：
Yuki Okawara;H. Hirano;Ayuko Kimura;N. Sato;Yuriko Hayashi;M. Osada;T. Kawakami;Norihisa Ootake;E. Kinoshita;Kiyotaka Fujita
PHOS-Tag对角线电泳法是为了准确鉴定PHOS-Tag-SDS-PAGE中磷蛋白电泳率的变化而开发的。以前，如果检测到单一的蛋白条带，就不可能确定在PHOS-Tag SDS-PAGE凝胶中Mn2+PHOS-Tag是否改变了蛋白质的迁移率，因为在不同的凝胶上进行了SDS-PAGE和PHOS-Tag SDS-PAGE。此外，当检测到多条蛋白质条带时，很难确定迁移率最高的条带是否被Mn2+PHOS-Tag改变了迁移率。然而，这些问题通过PHOS-Tag对角电泳法得到了解决，在该方法中，在单一凝胶上提供了SDS-PAGE和PHOS-Tag SDS-PAGE图谱。利用这项技术，我们鉴定了各种蛋白质的磷酸化状态，如α-乳清蛋白、α-和β-酪蛋白、卵清蛋白、碱性7S球蛋白和26S蛋白酶体亚基。在对人和酵母的26S蛋白酶体亚基的分析中，我们可以证实所有的亚基都是磷酸化的，并且发现尽管有许多磷酸化位点，但每个亚基中具有不同磷酸化状态的主要蛋白质的数量很少。然而，我们在这项技术中遇到了一个问题：当我们分别使用SDS-PAGE和PHOS-TAG SDS-PAGE时，通常很难识别Mn2+PHOS-Tag引起的迁移率变化。这样的问题通过PHOS-Tag对角电泳法得到了解决，在该方法中，在单个凝胶上提供了SDS-PAGE和PHOS-Tag-SDS-PAGE图谱。这项技术可用于鉴定各种蛋白质的磷酸化状态。例如：磷酸-标签对角线电泳法、质谱仪、磷蛋白、碱性7S球蛋白、蛋白酶体。
Phos-tag diagonal electrophoresis was developed to identify precisely a change in electrophoretic mobility of phosphoproteins in Phos-tag SDS-PAGE. Previously, if a single protein band was detected, it was impossible to determine whether mobility of the protein altered by Mn2+Phos-tag in Phos-tag SDS-PAGE gels because SDS-PAGE and Phos-tag SDS-PAGE were performed on different gels. Moreover, when multiple protein bands were detected, it was difficult to determine whether the band with the highest mobility was altered mobility by Mn2+Phos-tag. However, these problems were resolved by Phos-tag diagonal electrophoresis in which SDS-PAGE and Phos-tag SDS-PAGE patterns were provided on a single gel. Using this technique we identified phosphorylation states of various proteins such as α-lactalbumin, α- and β-casein, ovalbumin, basic 7S globulin, and 26S proteasome subunits. In the analyses of 26S proteasome subunits from humans and yeast, we could confirm that all subunits are phosphorylated, and find that the number of major proteins with different phosphorylation states is a few in each of the subunits despite having many phosphorylation sites.SignificancePreviously, Phos-tag SDS-PAGE has been developed to identify a change in electrophoretic mobility of phosphoproteins. However, we had a problem in this technique; it was often difficult to recognize the mobility shift by Mn2+Phos-tag when we used separately SDS-PAGE and Phos-tag SDS-PAGE. Such a problem was resolved by Phos-tag diagonal electrophoresis in which SDS-PAGE and Phos-tag SDS-PAGE patterns are provided on a single gel. This technique was useful to identify phosphorylation states of various proteins.:Phos-tag diagonal electrophoresis, mass spectrometry, phosphoproteins, basic 7S globulin, proteasome