Evaluation of protein phosphorylation state by a combination of vertical slab gel isoelectric focusing and immunoblotting.
Evaluation of protein phosphorylation state by a combination of vertical slab gel isoelectric focusing and immunoblotting.
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通过垂直平板凝胶等电聚焦和免疫印迹相结合评估蛋白质磷酸化状态。
DOI：
10.1016/0003-2697(89)90182-6
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发表时间：
1989
影响因子：
2.9
通讯作者：
Jagus,R
中科院分区：
文献类型：
--
作者：
Maurides,PA;Akkaraju,GR;Jagus,R
已经建立了使用垂直平板凝胶电泳的一维等电聚焦的条件，然后进行免疫印迹，用于测量蛋白质的磷酸化状态。该方法提供了一种耗时较少的替代二维凝胶电泳结合放射性标记或免疫印迹。该方法的主要优点是可以同时分析多个样品。该技术在这里适用于蛋白质合成的哺乳动物起始因子，真核起始因子2（eIF-2）的研究。当用于分析纯化的eIF-2或复杂混合物中所含的eIF-2时，该方法可以很好地分离和定量eIF-2 α亚基的不同磷酸化形式。该方法被证明是非常适合于测量快速磷酸化/去磷酸化动力学在细胞提取物中，以及在培养的细胞中的eIF-2的磷酸化状态的测量。此外，该方法证实了eIF-2上存在第二个磷酸化位点。虽然eIF-2已被用于证明该方法的有效性，但该技术适用于任何多肽的共价修饰的调节的研究，其中抗体是可用的。
Conditions have been established for one-dimensional isoelectric focusing using vertical slab gel electrophoresis, followed by immunoblotting, for the measurement of the phosphorylation state of proteins. The method provides a less time-consuming alternative to two-dimensional gel electrophoresis combined with radiolabeling or immunoblotting. The main advantage of the method is that many samples can be analyzed simultaneously. The technique is applied here to the study of a mammalian initiation factor for protein synthesis, eukaryotic initiation factor 2 (eIF-2). The method allows good separation and quantitation of the different phosphorylated forms of the α subunit of eIF-2, when used to analyze either purified eIF-2 or eIF-2 contained in complex mixtures. The method is shown to be well adapted to the measurement of rapid phosphorylation/dephosphorylation kinetics in cell extracts, as well as the measurement of the phosphorylation state of eIF-2 in cultured cells. In addition, the method is shown to confirm the existence of a second phosphorylation site on eIF-2. Although eIF-2 has been used for this demonstration of the efficacy of the method, the technique is applicable to a study of the regulation of covalent modification of any polypeptide for which antibodies are available.